Methods and compositions for treatment of inflammatory disease using cadherin-11 modulating agents

ABSTRACT

A method for treating inflammatory joint diseases by inhibiting cadherin-11 mediated cellular function using a cadherin-11 modulating agent is provided. Also provided are screening assays for identifying pharmaceutical lead compounds capable of modulating cellular functions of cadherin-11 such as cell proliferation, apoptosis, factor secretion, and binding of cadherin-11 to cadherin-11 counter-receptor inhibiting binding of cadherin-11 to its counter-receptor either in the context of a cell or in soluble form.

RELATED APPLICATIONS

This application is a divisional of U.S. Non-Provisional PatentApplication filed Aug. 31, 2005 now U.S. Pat. No. 7,488,478, entitled“METHODS AND COMPOSITIONS FOR TREATMENT OF INFLAMMATORY DISEASE USINGCADHERIN-11 MODULATING AGENTS”, Ser. No. 11/220,396, which is acontinuation of U.S. Non-Provisional Patent Application filed Jun. 9,2003, entitled “METHODS AND COMPOSITIONS FOR TREATMENT OF INFLAMMATORYDISEASE USING CADHERIN-11 MODULATING AGENTS”, Ser. No. 10/457,257, nowissued as U.S. Pat. No. 6,964,768, which is a divisional of U.S.Non-Provisional Patent Application filed Sep. 1, 2000, entitled “METHODSAND COMPOSITIONS FOR TREATMENT OF INFLAMMATORY DISEASE USING CADHERIN-11MODULATING AGENTS”, Ser. No. 09/654,328, now issued as U.S. Pat. No.6,787,136, which claims priority to U.S. Provisional Patent Applicationfiled Sep. 3, 1999, entitled “METHODS AND COMPOSITIONS FOR TREATMENT OFINFLAMMATORY DISEASE USING CADHERIN-11 INHIBITORY AGENTS”, Ser. No.60/152,456, now expired, and U.S. Provisional Patent Application filedSep. 13, 1999, entitled “METHODS AND COMPOSITIONS FOR TREATMENT OFINFLAMMATORY DISEASE USING CADHERIN-11INHIBITORY AGENTS”, Ser. No.60/153,490, now expired, the entire contents of all of which areincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made in part with government support under grantnumber AR34662 from the National Institutes of Health. The Governmentmay retain certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to methods and compositions for the treatment ofinflammatory joint disorders, such as those involving synovialhyperplasia and overproduction of biologically active factors. Themethods involve administering a cadherin-11 modulating agent to asubject to modulate cadherin-11 function in areas of joint damage.Screening assays for the identification of cadherin-11 modulating agentsare also provided.

BACKGROUND OF THE INVENTION

The adhesive interactions between cells and between cells and theextracellular matrix are believed to play critical roles in a widevariety of processes including, for example, modulation of the immunesystem, regulation of developmental processes and tumor progression andmetastasis. These interactions are mediated by adhesion molecules whichtransduce information from the extracellular to the intracellularmatrix.

Four families of adhesion molecules which mediate these interactionshave been identified: the integrins, the cadherins, the selectins, andimmunoglobulin-related molecules. In general, adhesion molecules aretransmembrane proteins which contain an extracellular domain forinteracting with an extracellular matrix or cellular component, atransmembrane domain spanning the cell membrane and a cytoplasmic domainfor interacting with one or more cytoskeletal or cytoplasmic components.

The cadherins play an important role in the establishment andmaintenance of intercellular connections between cells of the same type(reviewed in Geiger B. et al. (1992) Annual Review of Cell Biology8:307; Kemler R. (1993) Trends in Gastroenterology 9:317; Takeichi M.(1990) Annual Review of Biochem. 59:237; Takeichi M. (1991) Science251:1451). Cadherins are a superfamily of structurally related moleculesthat function in Ca⁺²-dependent homophilic adhesion. Cadherins areexpressed on cells that form solid tissues, and are responsible forsegregating and sorting cells during embryogenesis, establishing cellpolarity, and maintaining tissue morphology. Structurally, cadherins aresingle chain polypeptides that are synthesized as precursors and cleavedduring post-translational processing. They have large extracellularregions made up of 5 homologous domains, a single transmembrane segmentand a cytoplasmic tail.

The cadherins are synthesized as precursors that are cleaved duringpost-translational processing. The mature cadherins are single chainmolecules which include a relatively large extracellular domain(typically divided into five sections or “ectodomains”), a singletransmembrane region and a cytoplasmic tail. Among the classicalcadherins (i.e., P—(placenta), E—(epithelial), and N—(neural) cadherin),the cytoplasmic domain contains the highest degree of homology. The highdegree of homology observed for the cytoplasmic domain reportedly is areflection of the association of cadherins with a group of intracellularproteins, called catenins, that stabilize cadherin active conformation(Kemler R. (1993) Trends in Gastroenterology 9:317). It is generallybelieved that sequences in the extracellular domain are necessary tomediate homophilic (i.e., cadherin-to-cadherin) binding. A review of theliterature indicates that research directed to understandingcadherin-mediated adhesion has focussed on efforts to elucidate themechanism underlying cadherin-mediated homophilic cell adhesion. Littleattention has been directed to understanding what, if any, role isplayed by cadherins in heterophilic adhesion. While it has been knownfor some time that integrins and other adhesion molecules function inimmune system modulation, e.g., by playing a role in the adhesion ofperipheral lymphocytes to endothelium and in homing to lymph nodes,relatively little is known regarding the mechanism by which lymphocyteshome and transmigrate through the vascular endothelium to specificallytarget certain tissue locations, such as the synovium.

The most highly conserved sequence shared by cadherins lies within thecytoplasmic domain. It is this region that mediates interaction with thecytoplasmic catenins proteins (Hirano S. et al. Cell 70:293-301, 1992).The presence of the cytoplasmic domain is essential to functioning ofthe cadherin as deletions in this region abolish catenin binding as wellas cell-to-cell adhesion (Hulsken J, et al. J Cell Biol 127:1375-80,1994). The catenins (α, 102 kDa; β, 88-93 kDa, and γ, 80-83 kDa) beginto associate with cadherins almost immediately upon biosynthesis in astable manner that is not disrupted in TX-100 detergent (Takeichi M.Curr Opin Cell Biol 7:619-27, 1995) and are thought to mediate anchorageof cadherins to the. cytoskeleton (Yap A S. et al. Annu Rev Cell DevBiol 13:11946, 1997). Functionally, α-catenin is necessary for cadherinmediated homophilic adhesion. Tumor cells expressing E-cadherin at thecell surface, but lacking α-catenin expression, fail to formcell-to-cell contacts unless α-catenin expression is restored throughtransfection (Chen H. et al. J Cell Sci 1141345-56, 1997; and Knudsen KA. et al. J Cell Biol 130:67-77, 1995). β-catenin is homologous to theDrosophila segment polarity protein armadillo as well as the cadherinassociated protein plakoglobin. Plakoglobin, also termed y-catenin,interacts more weakly with the cadherin/catenin complex, and is notalways seen in cadherin precipitates.

A number of newly identified cadherin cDNA clones have been isolatedusing consensus oligonucleotides corresponding to cytoplasmic domainsequences that are highly conserved among cadherins and PCR cloning(Suzuki S. et al. Cell Reg 2:261-70, 1991).

Although cadherin function classically involves homophilic cell-to-celladhesion (i.e., E-cadherin binds E-cadherin typically on another cell ofthe same type), however, murine E-cadherin expressed on epidermalkeratinocytes also mediates adhesion to E-cadherin of Langerhans cells(Tang A. et al. Nature 361:82-5, 1993). Recently, we identified anothercounter-receptor for E-cadherin, namely the integrin α^(E)β₇ which isexpressed on intraepithelial T cells (Cepek K L. et al. Nature372:190-3, 1994 and U.S. Pat. No. 5,610,281). This represents an exampleof heterophilic binding of E-cadherin to the α^(E)β₇ integrincounter-receptor.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease ofunknown etiology that affects 1-2% of the population. It is dominated byprogressive joint destruction that can result in marked disability.Histologically, the joints reveal synovial hyperplasia mainly of type Asynoviocytes but also of type B synoviocytes, lymphocellularinfiltration of T and B cells, and neovascularization. These processeslead to the secretion of destructive factors and invasive growth of thesynovial membrane (pannus) into the adjacent cartilage and bone with theconsequent destruction of the joint.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery of cadherin-11 mRNAand protein expression in cells, namely human type B synoviocytes,recovered from the synovium of patients with inflammatory jointdisorders. Prior to the present discovery, cadherin-11 expression hadnot been reported in the synovium. Although not intending to be bound byany particular theory, it is postulated that cadherin-11 expression incells of the synovium is involved in the homing, retention andactivation of cells (such as T and B cells) in this area. In addition,cadherin-11 mediated adhesion is also postulated to mediate invasion ofsynovium into cartilage and bone during some inflammatory jointdisorders. Synoviocyte-synoviocyte contact may also be involved in someof these disorders. Cadherin-11 mediated adhesion can be characterizedas homophilic (cadherin-11 binding to cadherin-11) or heterophilic(cadherin-11 binding to a counter-receptor which is not cadherin-11).Accordingly, some of the compositions and methods of the presentinvention are directed towards inhibiting cadherin-11 mediated adhesionoccurring between these and other cell types. Still other methods andcompositions provided by the invention involve modulation of cellularfunctions which are mediated by cadherin-11, such as, for example, cellsignaling, proliferation, apoptosis and factor secretion.

According to one aspect on the invention, a method is provided fortreating a subject having an inflammatory joint disorder. The methodinvolves administering to a subject in need of such treatment atherapeutically effective amount of a cadherin-11 modulating agent. Acadherin-11 modulating agent is an agent that modulates (e.g., enhances,inhibits, changes, etc.) a cadherin-11 function. In one particularembodiment, the cadherin-11 modulating agent is a cadherin-11 inhibitoryagent. A cadherin-11 inhibitory agent is an agent which inhibits thebinding of cadherin-11 to a cadherin-11 counter-receptor. In preferredembodiments, the subject is a human. In more preferred embodiments, thesubject does not have abnormal cadherin-11 mediated adhesion occurringin the liver or the brain. Cadherin-11 mediated adhesion is the bindingof cadherin-11 to its counter-receptor.

In one embodiment, the inflammatory joint disorder is chronic synovitis.In another embodiment, the inflammatory joint disorder is an autoimmunedisease. In a preferred embodiment, the autoimmune disease is rheumatoidarthritis.

The cadherin-11 inhibitory agent may be administered systemically. Inpreferred embodiments, the cadherin-11 inhibitory agent is administeredlocally to a synovium or the synovial fluid of a subject.

The cadherin-11 inhibitory agent can inhibit cadherin-11 binding to acadherin-11 counter-receptor in a number of ways. In one embodiment, thecadherin-11 inhibitory agent binds selectively to cadherin-11. Inanother embodiment, the cadherin-11 inhibitory agent binds selectivelyto a cadherin-11 counter-receptor. Exemplary inhibitory agents aredescribed in the detailed description. In all of these embodiments, thecadherin-11 inhibitory agent functions to inhibit cadherin-11 mediatedadhesion.

The cadherin-11 and the cadherin-11 counter-receptor may be expressed bythe same or different cell types. The cadherin-11 counter-receptor neednot be on a cell surface but rather may be part of an interstitialmaterial or it may be a secreted material that binds to any othermolecule or surface. As an example of the latter embodiment, thecadherin-11 counter-receptor is a component of an extracellular matrixof a tissue, a cartilage or a bone. The cadherin-11 counter-receptor mayalso be a molecule secreted by a cell. In certain embodiments,cadherin-11 is expressed by one cell and the cadherin-11counter-receptor is expressed by another, distinct cell. Cadherin-11expressing cells may be selected from the group consisting of a type Aor a type B synoviocyte, a synovial derived fibroblast, a synovialmembrane lining cell, an osteoblast, a cartilage-derived cell and aninvasive pannus-derived cell. Cadherin-11 counter-receptor expressingcells may be selected from the group consisting of a T lymphocyte, a Blymphocyte, a plasma cell, a macrophage, a dendritic cell, a naturalkiller (NK) cell, a mast cell, a type A or a type B synoviocyte, asynovial derived fibroblast, an osteoblast, a cartilage-derived cell, asynovial membrane lining cell and an invasive pannus-derived cell.

In some embodiments, the cadherin-11 modulating agents, such as forexample, the cadherin-11 inhibitory agents are not antibodies, such asfor example monoclonal antibodies. In some embodiments, the agents ofthe invention do not include the antibodies disclosed in U.S. Pat. No.5,597,725 and in PCT application PCT/US93/03681 (WO/93/21302). Thus, incertain related embodiments, the agents of the invention do not embracethe monoclonal antibodies produced by the hybridomas designated 30Q8A(HB11316), 30Q4H (HB11317), 45A5G (HB 11318),30S2F (HB 11319),45C6A (HB11320),30T11G (HB 11324),64G11F (HB 11527).

The invention provides in another aspect a method for screening amolecular library to identify an agent (e.g., a pharmaceutical leadcompound) that modulates cadherin-11 mediated adhesion between a firstcell that expresses cadherin-11 and a second cell that expresses acadherin-11 counter-receptor. The agent may inhibit the cadherin-11mediated adhesion, in which case, it is a cadherin-11 inhibitory agent.The method involves performing a first adhesion assay between the firstcell and the second cell to obtain a first adhesion assay result,performing a second adhesion assay between the first cell and the secondcell in the presence of at least one molecular library member to obtaina second adhesion assay result, and comparing the first and the secondadhesion assay results to determine whether the at least one molecularlibrary member modulates cadherin-11 mediated adhesion between the firstcell and the second cell. The cell types, cadherin molecules andinhibitory agents are as described above and in the detaileddescription.

According to one embodiment, the first cell is selected from the groupconsisting of a synoviocyte such as a type A synoviocyte, a type Bsynoviocyte, a synovial derived fibroblast, a synovial membrane liningcell and an osteoblast. The second cell may be selected from the groupconsisting of a type A synoviocyte, a type B synoviocyte, a synovialderived fibroblast, a synovial membrane lining cell, an osteoblast, a Tlymphocyte, a B lymphocyte, a plasma cell, a macrophage, a dendriticcell, a natural killer cell and a mast cell. In yet another embodiment,the first cell is derived from the invasive pannus and the second cellis derived from cartilage. In a further embodiment, the first cell isderived from the invasive pannus and the second cell is an osteoblast.

According to the screening method provided, a difference in the firstand the second adhesion assay results is indicative of the presence ofat least one molecular library member that modulates cadherin-11mediated adhesion between the first cell and the second cell. The assaycan be designed to identify agents which inhibit or enhance thecadherin-11 mediated adhesion.

The molecular library may be recombinantly produced or chemicallysynthesized. In still other embodiments, the molecular library is apeptide library.

In yet another aspect of the invention, another method for screening amolecular library to identify a pharmaceutical lead compound thatmodulates cadherin-11 mediated adhesion is provided. The method involvesperforming a first adhesion assay between cadherin-11 and a cadherin-11counter-receptor to obtain a first adhesion assay result, performing asecond adhesion assay between cadherin-11 and the cadherin-11counter-receptor in the presence of at least one molecular librarymember to obtain a second adhesion assay result, and comparing the firstand the second adhesion assay results to determine whether the at leastone molecular library member modulates cadherin-11 mediated adhesion.

In certain embodiments of the invention, the cadherin-11counter-receptor is selected from the group consisting of a cadherin, anintegrin, an integrin subunit, an immunoglobulin family member and acarbohydrate. In preferred embodiments, the cadherin is cadherin-11. Inone embodiment, the cadherin-11 counter-receptor is a cadherin-11 fusionpolypeptide. In still another embodiment, the cadherin-11counter-receptor is an antibody that binds selectively to cadherin-11.

The cadherin-11 and/or the cadherin-11 counter-receptor may be isolated.The cadherin-11 and/or the cadherin-11 counter-receptor may also besoluble.

In yet another embodiment, cadherin-11 is presented by a cell. In yet afurther embodiment, the cadherin-11 counter-receptor is presented by acell. The cell expressing cadherin-11 may be selected from the groupconsisting of a type A or a type B synoviocyte, a synovial derivedfibroblast, a synovial membrane lining cell, an osteoblast, acartilage-derived cell and an invasive pannus-derived cell. The cellexpressing cadherin-11 counter-receptor may be selected from the groupconsisting of a synoviocyte, a synovial derived fibroblast, a synovialmembrane lining cell, an osteoblast, a cartilage-derived cell, aninvasive pannus-derived cell, a T lymphocyte, a B lymphocyte, a plasmacell, a macrophage, a dendritic cell, a mast cell and a natural killercell. In still other embodiments, the cadherin-11 counter-receptor maybe a component of an extracellular matrix of tissue, cartilage or bone,or it may be a molecule secreted by a cell and found anywhere in atissue.

In another aspect, the invention provides a method for treating asubject having an inflammatory joint disorder comprising administeringto a subject in need of such treatment a therapeutically effectiveamount of an agent which modulates a cellular function in a cadherin-11expressing cell, preferably a cellular function other than cadherin-11mediated adhesion. The agent may modulate the activity by interactingdirectly with cadherin-11 (e.g., a binding agent which selectively bindsto cadherin-11 and thereby modulates its activity) or indirectly byinteracting with another cellular component which then modulates thecadherin-11 activity. The cellular function may be selected from thegroup consisting of cell proliferation, factor secretion, apoptosis,migration, and/or attachment. In important embodiments, the agent whichmodulates a cellular function in a cadherin-11 expressing cell does notinhibit binding of cadherin-11 to a cadherin-11 counter-receptor. Thus,in some embodiments, the cellular function is not cadherin-11 binding toa cadherin-11 counter-receptor (i.e., the cellular function is notcadherin-11 mediated adhesion).

In yet another aspect of the invention, a method for screening amolecular library to identify a pharmaceutical lead compound thatmodulates a cellular function in a cadherin-11 expressing cell(preferably, a cellular function other than cadherin-11 mediatedadhesion) is provided. The method comprises determining a first value ofthe cellular function for a cadherin-11 expressing cell in the absenceof a molecular library member, determining a second value of thecellular function for a cadherin-11 expressing cell in the presence ofat least one molecular library member, and comparing the first value andthe second value to determine whether the at least one molecular librarymember modulates a cellular function in a cadherin-11 expressing cell.The cellular function may be selected from the group consisting of cellproliferation, factor secretion, apoptosis, migration and/or attachment.

In a related aspect, the invention provides a similar method forscreening a molecular library to identify a pharmaceutical lead compoundthat modulates factor secretion in a cadherin-11 expressing cell. In oneembodiment of this latter aspect, the factor secretion is selected fromthe group consisting of the factor secretion is selected from the groupconsisting of stromelysin secretion, collagen secretion, collagenasesecretion and IL-6 secretion.

In a similar manner, the screening method can be used to identifypharmaceutical lead compounds that modulate a cellular function in acadherin-11 counter receptor expressing cell. In important embodiments,the pharmaceutical lead compound is screened for its ability to inhibitcadherin-11 binding to a cadherin-11 counter-receptor. Preferably, incertain embodiments, the pharmaceutical lead compound does not inhibitbinding of cadherin-11 to its counter-receptor. Thus in someembodiments, the cellular function which is modulated is not cadherin-11binding to a cadherin-11 counter-receptor.

The invention also provides, in another aspect, a pharmaceuticalcomposition (i.e., a pharmaceutical preparation) comprising an effectiveamount of a cadherin-11 modulating agent (e.g., a cadherin-11 inhibitoryagent) and a pharmaceutically acceptable carrier as well as a method formaking such a composition. The cadherin-11 modulating agent is presentin an amount effective to modulate cadherin-11 function. If the agent isa cadherin-11 inhibitory agent, then it may be present in an amounteffective to inhibit cadherin-11 mediated adhesion (such as, forexample, that which occurs in the synovium). In one embodiment, thecomposition is contained in a syringe for injection locally to thesynovium, synovial fluid, joint or joint capsule of a subject.

The invention further provides a method of making a pharmaceuticalcomposition, a pharmaceutical preparation and/or a medicament, i.e., byplacing the cadherin-11 modulating agent (e.g., a cadherin-11 inhibitoryagent) of the invention in a pharmaceutically acceptable carrier. Thepharmaceutical composition preparation may contain one or morecadherin-11 modulating agents and, optionally, other therapeutic agentswhich are useful in the treatment of disorder described herein (e.g.,NSAIDs).

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent with reference to the detaileddescription of the preferred embodiments and to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The Examples refer to and include a brief description of variousfigures. It is to be understood that the drawings or figures areillustrative only and are not required for the enablement of theinventions disclosed herein.

FIG. 1 illustrates gel electrophoresis separated PCR products. Thenegative control is shown in lane 1 and the expected 385 bp size productobtained in type B synoviocytes cDNA is shown in lane 2.

FIG. 2 illustrates results from a Northern analysis of mRNA from 16E6.A5EpC line (lane 1), type B synoviocytes (lane 2), Jurkat cells (lane 3)and the neuroblastoma cell line SKNSH (lane 4). hybridized with the 385bp fragment of cadherin-11 (top panel) and a glyceraldehyde-3-phosphatedehydrogenase probe (Clontech) (bottom panel) as a control. Themolecular mass markers is indicated on the left.

FIG. 3 is a schematic of the structure of the human cadherin-11-Fcfusion protein. The sequence of the extracellular juxtamembrane regionof wild-type cadherin-11 (nucleic acid sequence is SEQ ID NO:13; aminoacid sequence is SEQ ID NO:14) and the alterations resulting from fusionwith the human Fc region (nucleic acid sequence is SEQ ID NO:15; aminoacid sequence is SEQ ID NO:16) are shown. Regions corresponding to theFc portion are shown in bold.

FIG. 4 is a series of flow cytometry histograms showing cell surfaceexpression of cadherin-11 in L-cells transfected with cadherin-11 cDNA.L-cells transfected with pLK-neo (left column) or with pLK-neo/C11(right column) are shown. Staining with control mAb P3 is shownunshaded. Staining with αCad-11, 3H10, 5H6, and 2G4 is shown shaded.

FIG. 5 is a photocopy of a photograph illustrating theimmunohistochemistry staining of synovial lining cells and somesublining cells in RA synovitis using mAb 2G4. Frozen tissue sections ofhuman RA synovium (panel A and B) were stained by the indirectimmunoperoxidase method using 2G4 mAb and counterstained withhematoxylin (magnification 20×[panel A] and 60×[panel B]). Most of thelining cells and few in the sublining were 2G4+ (panel B).

FIG. 6 illustrates a flow cytometric analysis of cadherin-11 expressionby synovial T cell line 5 (left panel) and CP-B cells (right panel).

FIG. 7 is a plot of the percent of synovial T cell line 5 cells and CP-Bcells bound to control proteins or cadherin-11.

SEQUENCE LISTING

SEQ ID NO: 1 is the nucleotide sequence of human cadherin-11 cDNA.

SEQ ID NO: 2 is the amino acid sequence of human cadherin-11 protein.

SEQ ID NO: 3 is the amino acid sequence of human E-cadherin residues753-762.

SEQ ID NO: 4 is the amino acid sequence of human E-cadherin residues840-847.

SEQ ID NO: 5 is the amino acid sequence of human E-cadherin residues853-859.

SEQ ID NO: 6 is the amino acid sequence of human E-cadherin residues865-875.

SEQ ID NO: 7 is the nucleotide sequence of a degenerate sense primer.

SEQ ID NO: 8 is the nucleotide sequence of a degenerate antisenseprimer.

SEQ ID NO: 9 is the nucleotide sequence of primer XV14.

SEQ ID NO: 10 is the nucleotide sequence of primer XV15.

SEQ ID NO: 11 is the nucleotide sequence of primer XVCad11A.

SEQ ID NO: 12 is the nucleotide sequence of primer XVCad11E.

DETAILED DESCRIPTION OF THE INVENTION

The invention is premised, in part, on the discovery that cadherin-11mRNA and protein are expressed in cells of the synovium of patients withinflammatory joint disorders. The discovery was made by co-precipitatingcadherin-11 with an antiserum to catenin in type B human synoviocytes.The demonstration of expression of a cadherin, namely cadherin-11, inthe synovium of a rheumatoid arthritis patient provides a previouslyunrecognized opportunity to target therapies at ameliorating rheumatoidarthritis as well as other inflammatory arthritis in which synovialhyperplasia and overproduction of toxic and biologically active factorsmediate joint damage.

Cadherin-11 is a transmembrane molecule that, inter alia, mediatesbinding of cells to each other through interaction with itself or itscounter-receptors. Like other cadherins, cadherin-11 is proposed tomediate adhesion of like cells to each other as well as adhesion ofcells of different lineages to each other. According to the discoveryupon which the present invention is based, cadherin-11 is proposed,inter alia, to mediate adhesion between like cells such as synoviocytesas well as different cells such as synoviocytes and lymphocytes (e.g., Tand B cells).

The human and mouse cadherin-11 genes have been isolated and sequencedpreviously (Suzuki S. et al. Cell Reg 2:261-70, 1991). See also, GenbankAccession No. NM_(—)001797, (SEQ ID NO: 1 and SEQ ID NO: 2) for thehuman cadherin-11 cDNA and predicted amino acid sequences, respectively.

Lymphocytes have the ability to circulate through blood vessels and theneither sample antigens or provide immune protection in all tissues.Specific adhesion molecules (e.g., selectins and integrins) and theircounter-receptors are believed to mediate lymphocyte homing andtransmigration through the vascular endothelium at sites of inflammation(Springer T A. Cell 76:301-314, 1994). Each integrin is a noncovalentlyassociated αβ heterodimeric complex. Integrin subfamilies have beendefined by the use of particular β chains which often pair with one ofseveral different α chains. Only selected integrins are expressed on Tcells. Members of the β₁ and β₃ subfamily (α¹β₁, α²β₁, α⁴β₁α⁵β₁ andα^(v)β₃) are primarily involved in lymphocyte adhesion to extracellularmatrix, except for the α⁴β₁, heterodimer (VLA-4) that binds to vascularcell adhesion molecule-1 (VCAM-1), a counter-receptor on activatedvascular endothelium (Elices M J. et al. Cell 60:577-584, 1990). Of theα subfamily, LFA-1 (α^(L)β₂) is expressed on T cells and mediatesbinding to counter-receptors ICAM-1 (Marlin S D. et al. Cell 51:813-819,1987), ICAM-2 (deFourgerolles A R. et al. J Exp Med 174:253-267, 1991)and ICAM-3 (deFourgerolles A R. et al. J Exp Med 175:185-190, 1992;Fawcett J. et al. Nature 360:481-484, 1992; and Vazeux R. et al. Nature1992; 360:481-488). LFA-1 also mediates attachment to inflamed vascularendothelium allowing firm attachment of T lymphocytes prior to theirtransmigration out of the blood-stream. The migration of lymphocytesinto inflammatory sites involves a multi-step process in which selectins(such as L-selectin on T-cells) mediate rolling, following whichintegrin activation results in LFA-1 and VLA-4 mediated tightattachment, followed by transmigration into tissues. This trafficking oflymphocytes is a requirement for lymphocyte accumulation at sites ofinfection, or abnormally at sites of chronic inflammation such asrheumatoid synovium.

Thus, the invention embraces the use of cadherin-11 modulating agents inthe treatment of inflammatory joint disorders. Additionally, screeningmethods are provided for the identification of cadherin-11 modulatingagents which possess the characteristics described herein.

The methods and compositions of the invention relate to cadherin-11modulating agents. A cadherin-11 modulating agent, as used herein,embraces agents which inhibit the binding of cadherin-11 to itscounter-receptor (i.e., cadherin-11 inhibitory agents) and agents whichmodulate cellular functions in cadherin-11 expressing cells (e.g.,agents which trigger, or those which inhibit, cadherin-11 associatedsignaling). Cadherin-11 modulating agents are able to modulate (1) cellproliferation, (2) secretion of molecules such as, but not limited to,stromelysin, collagen, collagenase and IL-6, (3) apoptosis, (4)migration and/or (5) attachment, of cadherin-11 expressing cells. Inimportant embodiments, such agents lead to a decrease in cellproliferation, a decrease in the secretion of molecules such as, but notlimited to, cytokines, and an increase in apoptosis in cells expressingcadherin-11. In effect, these agents preferably function to slow therate of growth of, or ultimately kill, cadherin-11 cells such assynoviocytes. It has been reported previously in other cell types thatloss of E-cadherin may lead to a cancerous or metastatic phenotype.

The invention thus also embraces cadherin-11 inhibitory agents and theirmethod of use. Unless otherwise stated, it is to be understood thatthese two categories of agents, i.e., agents which modulate cadherin-11mediated adhesion (i.e., cadherin-11 inhibitory agents) and agents whichmodulate other cadherin-11 functions, such as proliferation, factorsecretion, and apoptosis, need not be mutually exclusive nor need theybe completely overlapping. For example, it is expected that some agentsidentified in the screening assays of the invention will function solelyas cadherin-11 inhibitory agents (i.e., having the ability to blockcadherin-11 binding to its counter-receptor), others will function bymodulating (e.g., enhancing or inhibiting) cellular functions, such asproliferation, secretion, apoptosis, attachment and migration, whilestill other agents will be capable of both. Thus, some agents will becapable of inhibiting cadherin-11 binding to its counter-receptor, yethave no impact upon other cadherin-11 mediated functions such as cellsignaling. Similarly, other agents will enhance or inhibit cadherin-11mediated functions such as proliferation, for example, yet have noeffect on cadherin-11 binding to its counter-receptor.

In one aspect, the invention is directed to a method for treating asubject having an inflammatory joint disorder. As used herein, aninflammatory joint disorder is one in which synovial hyperplasia and/oroverproduction of biologically active factors mediate joint damage. Ininflammatory joint disorders, the synovium becomes inflamed andthickened, and in advanced cases, this is followed by an invasion of thesynovium into the cartilage and bone. The synovium is the membranelining the capsule of a joint. Synovitis (i.e., inflammation of thesynovium) can be manifest in either acute or chronic forms. In acutesynovitis, the onset of pain and discomfort is usually sudden and ofshort duration, as is also the case in pigment villonodular synovitis.In contrast, in chronic synovitis, the pain and discomfort are recurrentand persistent. In either case, the symptoms can be brought on byarthritis, injury, overuse of the joint and infection.

The modulating agent, such as the inhibitory agent, is preferablyadministered locally to the synovium of the subject. Affected jointswhich can be treated are those which exist throughout the body, in areassuch as the shoulders, back, wrists, hands, elbows, knees, hips, anklesand feet, and are lined by a synovial membrane.

Exemplary conditions that result from or cause inflammatory jointdisorders include chronic synovitis, autoimmune disorders, psoriaticarthritis, chronic Lyme disease arthritis, arthritis associated withinflammatory bowel disease, arthritis associated with ankylosingspondylitis, Reiter's syndrome, arthritis associated with systemic lupuserythrematosus, arthritis associated with juvenile chronic arthritis,arthritis associated with infection, arthritis associated with immuneresponse to infectious agents and nonspecific synovitis of unknownetiology. An autoimmune disorder is one in which the body's immunesystem reacts against one or more body tissues and thus attacks thetissue as it would a foreign antigen or pathogen. Rheumatoid arthritisis an example of an autoimmune disorder in which the immune systemcomponents perpetuate inflammation of a joint, resulting in damage tothe synovium, cartilage, bone, and associated joint tissues.

One method provided by the invention involves administering to a subjectin need of such treatment a cadherin-11 inhibitory agent. A cadherin-11inhibitory agent is an agent which inhibits the binding of cadherin-11to a cadherin-11 counter-receptor. The cadherin-11 inhibitory agent isadministered to the subject in a therapeutically effective amount. Atherapeutically effective amount is a dosage of the cadherin-11inhibitory agent sufficient to provide a medically desirable result. Inthe treatment method of the invention, the therapeutically effectiveamount of the cadherin-11 inhibitory agent may be that amount which issufficient to reduce the inflammation or swelling of the joint, or toalleviate the pain at the joint. As used herein, treatment embraces theuse of the cadherin-11 inhibitory agents in reducing the adverse medicalcondition that is mediated by cadherin-11 binding to itscounter-receptor in vivo, as well as prophylactic treatment of subjectsat risk of developing an inflammatory joint disorder.

As mentioned earlier, the treatment methods described herein embrace theuse of either or both cadherin-11 inhibitory agents and agents whichmodulate cellular functions (e.g., signaling, proliferation, apoptosis,etc.) of cadherin-11 other than cadherin-11 binding to a cadherin-11counter-receptor. Thus, as described herein, the method of treatment ofa subject having an inflammatory joint disorder may alternativelycomprise administering to the subject an effective amount of an agentthat modulates one or more cellular functions of cadherin-11. Inimportant embodiments, the one or more cellular functions do not includecadherin-11 binding to its counter-receptor.

A subject, as used herein, refers to any mammal susceptible to having orpresently having an inflammatory joint disorder. Preferably, the subjectis one having an inflammatory joint disorder. In more preferredembodiments, the subject is a human. In certain embodiments, the subjectdoes not have abnormal cadherin-11 mediated adhesion in the brain and/orliver.

In general, cadherin-11 inhibitory agents are agents which inhibitcadherin-11 mediated adhesion. Cadherin-11 mediated adhesion, as usedherein, refers to the binding of a cadherin-11 polypeptide, herein afterreferred to as cadherin-11, to a cadherin-11 counter-receptor. Acadherin-11 counter-receptor is a molecule which selectively binds tocadherin-11 and, in some instances, is expressed at the surface of acell. In other instances, the cadherin-11 counter-receptor is notcell-bound but is rather a component of an interstitial matrix such asan extracellular matrix of tissue, cartilage or bone. The cadherin-11counter-receptor may also be a molecule secreted by a cell. Exemplarycells which express a cadherin-11 counter-receptor include type A andtype B synoviocytes, synovial-derived fibroblasts, cartilage-derivedcells, osteoblasts, T and B lymphocytes, plasma cells, macrophages,dendritic cells, NK cells, mast cells, synovial lining cells, and cellsderived from the invasive pannus. A cadherin-11 counter-receptorembraces cadherin-11, members of the immunoglobulin superfamily,carbohydrate epitopes of a glycoprotein or glycolipid, a selectin, alectin containing ligand and an integrin. At least 21 differentintegrins have been identified composed from eight different β chainsassociated with 15 different α chains. Integrins useful in the inventioninclude α¹β₁, α²β₁, α³β₁, α⁴β₁, α⁵β₁, α⁶β₁, α⁷β₁, α⁸β₁, α⁹β₁, α^(L)β₂,α^(M)β₂, α^(x)β₂, α^(IIb)β₃, α^(v)β₃, α⁶β₄, α^(v)β₅, α⁴β₇, α^(E)β₇,α⁶β₇. In some embodiments, the preferred integrins are α¹β₁, α²β₁, α³β₁,α⁴β₁, α⁵β₁, α⁶β₁, α^(L)β₂, α^(M)β₂, α^(v)β₃, α⁴β₇ and α^(E)β₇.

The invention is premised in part on the observation that T cells and Bcells, both of which have not been previously reported to expresscadherin-11, are capable of binding to immobilized recombinantcadherin-11 in vitro. Thus, this finding suggests that T and B cellsexpress a cadherin-11 counter-receptor which is not cadherin-11. This isthe first report of a cadherin-11 counter-receptor which is notcadherin-11 expressed by T and B cells. By comparing cells which bind topurified or isolated cadherin-11 (and which are known not to expresscadherin-11) to cells which are incapable of such binding, cadherin-11counter-receptors which are not cadherin-11 can be identified. Exemplarymethods for comparison of these two cell types include, e.g., geneticscreening using techniques such as differential display, as well as,subtractive hybridization to identify transcripts which are expressed bythe cadherin-11 binding cells and not expressed by the cadherin-11non-binding cells. These techniques are routinely practiced by those ofordinary skill in the art. These techniques allow for the rapidisolation of nucleic acid coding for a cadherin-11 counter-receptor.Once cells have been identified which bind cadherin-11 but do notexpress cadherin-11 are identified, cellular membrane preparations canbe analyzed by, for example, affinity purification to isolate thecadherin-11 counter-receptor. For example, a GST-cadherin-11 fusionprotein immobilized on a column can be used to extract cadherin-11counter-receptors from a crude cell preparation. In a preferredembodiment, monoclonal antibodies are generated against cells thatexpress a cadherin-11 counter-receptor and the resultant antibodies arescreened for their ability to block cadherin-11 binding to such cells.

The cadherin-11 modulating agents of the invention, including thecadherin-11 inhibitory agents, embrace nucleic acid molecules,polypeptides, carbohydrates, and synthetically-produced andrecombinantly-produced molecules.

In certain other embodiments of the invention, the cadherin-11modulating agent is a polypeptide (i.e., a cadherin-11 modulatingpolypeptide). As such, the modulating agent, including the cadherin-11inhibitory agent, may be (1) a cadherin-11 polypeptide or a fragment(preferably unique) thereof, or (2) a cadherin-11 counter-receptorpolypeptide or a fragment (preferably unique) thereof; (3) a cadherin-11“binding peptide” (other than a cadherin-11 counter-receptor); and (4) acadherin-11 counter-receptor “binding peptide” (other than a cadherin-11polypeptide). As used herein, a cadherin-11 binding peptide refers topeptides which bind selectively to cadherin-11. Similarly, a cadherin-11counter-receptor binding peptide refers to a peptide which bindsselectively to cadherin-11 counter-receptor. In some importantembodiments, the cadherin-11 binding peptide does not inhibit thebinding of cadherin-11 to a cadherin-11 counter-receptor. That is tosay, that in certain embodiments, the binding peptide is not acadherin-11 inhibitory agent, but it may still modulate othercadherin-11 mediated functions such as proliferation. The same may betrue for cadherin-11 counter-receptor binding peptides. The inventionembraces the use of these latter two classes of binding peptides in thetreatment of subjects where it is not necessary to inhibit cadherin-11binding to a cadherin-11 counter-receptor in order to effect treatment.

Binding peptides can be antibodies or fragments of antibodies (“bindingpolypeptides”), having the ability to bind selectively to cadherin-11 orcadherin-11 counter-receptor polypeptides. Antibodies include polyclonaland monoclonal antibodies which can be prepared according toconventional methodology. Binding peptides and binding polypeptides canalso be derived from sources other than antibody technology. Forexample, such binding peptides can be provided by degenerate peptidelibraries which can be readily prepared in solution, in immobilizedform, as bacterial flagella peptide display libraries or as phagedisplay libraries. Combinatorial libraries also can be synthesized ofpeptides containing one or more amino acids. Libraries further can besynthesized of peptides and non-peptide synthetic moieties. Exemplarycadherin-11 binding peptides which are antibodies and fusion proteinsare described in the Examples. (See also, U.S. Pat. Nos. 5,639,634 and5,597,725 and PCT Patent Application No. WO93/21302, the entire contentsof which are incorporated by reference herein.)

Cadherin-11 inhibitory polypeptides may be cell adhesion molecules suchas integrins and cadherins.

Cadherin-11 inhibitory polypeptides are useful for inhibiting thebinding of cadherin-11 to its counter-receptor. As used herein, afragment of a polypeptide refers to one which is capable of binding toeither cadherin-11 or the cadherin-11 counter-receptor, as the case maybe. The preferred cadherin-11 inhibitory polypeptides of the inventionhave the amino acid sequence of SEQ. ID NO. 2 or a functionallyequivalent fragment of SEQ. ID NO.2. Thus, cadherin-11 inhibitorypolypeptides embrace functionally equivalent fragments, variants, andanalogs of SEQ. ID NO. 2, provided that the fragments, variants, andanalogs bind to cadherin-11 or to a cadherin-11 counter-receptor and,thereby, reduce or prevent cadherin-11 adhesion to its counter-receptor.The invention also embraces proteins and peptides coded for by any ofthe foregoing cadherin-11 inhibitory nucleic acid molecules.

A unique fragment of a cadherin-11 polypeptide, in general, has thefeatures and characteristics of unique fragments as discussed herein inconnection with nucleic acids. As will be recognized by those skilled inthe art, the size of the unique fragment will depend upon factors suchas whether the fragment constitutes a portion of a conserved proteindomain. Thus, some regions of SEQ ID NO:2 will require longer segmentsto be unique while others will require only short segments, typicallybetween 5 and 12 amino acids (e.g. 5, 6, 7, 8, 9, 10, 11 and 12 aminoacids long or more, including each integer up to the full lengthpolypeptide). Virtually any segment of SEQ ID NO:2 that is 9 or moreamino acids in length and which is not common to other distinctpolypeptides will be unique. Unique fragments of a polypeptidepreferably are those fragments which retain a distinct functionalcapability of the polypeptide. Functional capabilities which can beretained in a unique fragment of the cadherin-11 polypeptide include theability to bind to a cadherin-11 counter-receptor. Similarly a uniquefragment of a cadherin-11 counter-receptor polypeptide will possess theability to bind to a cadherin-11 polypeptide.

As used herein with respect to polypeptides, the term “isolated” meansseparated from its native environment in sufficiently pure form so thatit can be manipulated or used for any one of the purposes of theinvention. Thus, isolated means sufficiently pure to be used (i) toraise and/or isolate antibodies, (ii) as a reagent in an assay, or (iii)for sequencing, etc.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fl, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe cadherin-11 or cadherin-11 counter-receptor polypeptide. Thisprocess can be repeated through several cycles of reselection of phagethat bind to the cadherin-11 or cadherin-11 counter-receptorpolypeptide. Repeated rounds lead to enrichment of phage bearingparticular sequences. The displayed peptide sequence can vary in size.As the size increases, the complexity of the library increases. It ispreferred that the total size of the displayed peptide sequence (therandom amino acids plus any spacer amino acids) should not be greaterthan about 100 amino acids long, more preferably not greater than about50 amino acids long, and most preferably not greater than about 25 aminoacids long. In certain embodiments, the libraries may have at least oneconstraint imposed upon the displayed peptide sequence. A constraintincludes, but is not limited to, a positive or negative charge,hydrophobicity, hydrophilicity, a cleavable bond and the necessaryresidues surrounding that bond, and combinations thereof. In certainembodiments, more than one constraint is present in each of the peptidesequences of the library.

DNA sequence analysis can be conducted to identify the sequences of theexpressed polypeptides. The minimal linear portion of the sequence thatbinds to the cadherin-11 or cadherin-11 counter-receptor polypeptide canbe determined. One can repeat the procedure using a biased librarycontaining inserts containing part or all of the minimal linear portionplus one or more additional degenerate residues upstream or downstreamthereof. Yeast two-hybrid screening methods also may be used to identifypolypeptides that bind to the cadherin-11 or cadherin-11counter-receptor polypeptides. Thus, the cadherin-11 or cadherin-11counter-receptor polypeptides of the invention, or a fragment thereof,can be used to screen peptide libraries, including phage displaylibraries, to identify and select peptide binding partners of thecadherin-11 or cadherin-11 counter-receptor polypeptides of theinvention. Such molecules can be used, as described, for screeningassays, for purification protocols, for interfering directly with thefunctioning of cadherin-11 or cadherin-11 counter-receptor and for otherpurposes that will be apparent to those of ordinary skill in the art.

A cadherin-11 or cadherin-11 counter-receptor polypeptide, or a fragmentthereof, also can be used to isolate other binding partners (e.g.,naturally occurring cadherin-11 counter-receptors). Isolation of bindingpartners may be performed according to well-known methods. For example,isolated cadherin-11 or cadherin-11 counter-receptor polypeptides can beattached to a substrate, and then a solution suspected of containing ancadherin-11 or cadherin-11 counter-receptor binding partner may beapplied to the substrate. If the binding partner for cadherin-11 orcadherin-11 counter-receptor polypeptides is present in the solution,then it will bind to the substrate-bound cadherin-11 or cadherin-11counter-receptor polypeptide. The binding partner then may be isolated.

In some important embodiments, the cadherin-11 inhibitory agent is afunctionally equivalent peptide analog of cadherin-11 or a cadherin-11counter-receptor. As used herein, the term functionally equivalentpeptide analog refers to a peptide analog that is capable of inhibitingthe binding of cadherin-11 to the cadherin-11 counter-receptor bycompeting either with cadherin-11 for binding to a counter-receptor ofcadherin-11 or with cadherin-11 counter-receptor for binding tocadherin-11. Functionally equivalent peptide analogs of cadherin-11 areidentified, for example, in in vitro adhesion assays, as described belowand in the Examples, that measure the ability of the peptide analog toinhibit cadherin-11-mediated adhesion either between cells expressingcadherin-11 and its counter-receptor or between isolated cadherin-11 andisolated cadherin-11 counter-receptor, or some combination thereof.Accordingly, exemplary functionally equivalent peptide analogs ofcadherin-11 include the extracellular domain of cadherin-11, fragmentsof the extracellular domain and peptide analogs of the extracellulardomain (e.g., peptides which contain conservative amino acidsubstitutions), provided that the peptide fragments and analogs arecapable of inhibiting adhesion between cadherin-11 and itscounter-receptor when these molecules are present in isolated form or inthe context of a cell membrane in vivo and/or in vitro. Likewise, afunctionally equivalent peptide analog of cadherin-11 counter-receptorincludes the extracellular domain of cadherin-11 counter-receptor,fragments of the extracellular domain and peptide analogs of theextracellular domain (e.g., peptides which contain conservative aminoacid substitutions), provided that the peptide fragments and analogs arecapable of inhibiting adhesion between cadherin-11 and itscounter-receptor when these molecules are present in isolated form or inthe context of a cell membrane in vivo and/or in vitro. Preferably, thepeptide fragments and/or analogs contain between about three and aboutone hundred amino acids. More preferably, the peptide analogs containbetween about ten and about twenty-five amino acids.

The inhibitory agents of the invention include antibodies and fusionproteins that inhibit binding of cadherin-11 to its counter-receptor.Thus, the peptides of the invention can be specifically reactive with anantibody (preferably a monoclonal antibody) that binds selectively tocadherin-11 or an antibody that binds selectively to cadherin-11counter-receptor and thereby inhibits adhesion between cadherin-11 and acounter-receptor of cadherin-11. Thus, fusion polypeptides ofcadherin-11 and cadherin-11 counter-receptor are also embraced by thepresent invention, as is their use in the methods disclosed herein. Afusion polypeptide, as used herein, is a polypeptide which containspeptide regions from at least two different proteins. For example, afusion cadherin-11 polypeptide can be formed by fusing, usually at thenucleotide level, coding sequence from cadherin-11 to coding sequencefrom another distinct protein. As described below, a cadherin-11 GSTfusion protein can be synthesized and is useful in screening for agentswhich bind to cadherin-11. Also as described in the Examples, acadherin-11-Fc fusion protein has been synthesized by joining theextracellular domains of cadherin-11 to the Fc portion ofimmunoglobulin. Depending on their nature, some fusion proteins aresuited for in vitro use while others are better suited to in vivo use.

In certain preferred embodiments, the cadherin-11 modulating agent is anantibody or a fragment of an antibody having the ability to selectivelybind to cadherin-11 or cadherin-11 counter-receptor polypeptides.Antibodies include polyclonal and monoclonal antibodies, preparedaccording to conventional methodology.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3).

The CDRs, and in particular the CDR3 regions, and more particularly theheavy chain CDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody.

Thus, for example, PCT International Publication Number WO 92/04381teaches the production and use of humanized murine RSV antibodies inwhich at least a portion of the murine FR regions have been replaced byFR regions of human origin. Such antibodies, including fragments ofintact antibodies with antigen-binding ability, are often referred to as“chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

For inoculation or prophylactic uses, the antibodies of the presentinvention are preferably intact antibody molecules including the Fcregion. Such intact antibodies will have longer half-lives than smallerfragment antibodies (e.g. Fab) and are more suitable for intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal administration.

Fab fragments, including chimeric Fab fragments, are preferred inmethods in which the peptides of the invention are administered directlyto a local tissue environment. For example, the Fab fragments arepreferred when the peptide of the invention is administered directly tothe afflicted joint or to the inflamed synovium. Fabs offer severaladvantages over F(ab′)₂ and whole immunoglobulin molecules for thistherapeutic modality. First, because Fabs have only one binding site fortheir cognate antigen, the formation of immune complexes is precludedwhereas such complexes can be generated when bivalent F(ab′)₂ and wholeimmunoglobulin molecules encounter their target antigen. This is of someimportance because immune complex deposition in tissues can produceadverse inflammatory reactions. Second, because Fabs lack an Fc regionthey cannot trigger adverse inflammatory reactions that are activated byFc, such as activation of the complement cascade. Third, the tissuepenetration of the small Fab molecule is likely to be much better thanthat of the larger whole antibody. Fourth, Fabs can be produced easilyand inexpensively in bacteria, such as E. coli, whereas wholeimmunoglobulin antibody molecules require mammalian cells for theirproduction in useful amounts. Production of Fabs in E. coli makes itpossible to produce these antibody fragments in large fermenters whichare less expensive than cell culture-derived products.

A cadherin-11 modulating agent may also be a nucleic acid molecule(i.e., a cadherin-11 modulating nucleic acid molecule). A cadherin-11modulating nucleic acid molecule is a nucleic acid molecule thatfunctions to modulate a cadherin-11 cellular function, such as forexample, cell proliferation, factor secretion, apoptosis, migration andattachment. A cadherin-11 inhibitory nucleic acid molecule is a nucleicacid molecule that functions to inhibit cadherin-11 mediated adhesion.These nucleic acid molecules may function directly (e.g., as antisensenucleic acid molecules) or indirectly (e.g., via the peptides and/orpolypeptides they encode). A cadherin-11 modulating nucleic acidmolecule and a cadherin-11 inhibitory nucleic acid molecule may beselected from a group consisting of nucleic acid molecules which (1)encode a cadherin-11 polypeptide or a fragment (preferably unique)thereof; (2) encode a cadherin-11 counter-receptor polypeptide or afragment (preferably unique) thereof, or (3) are cadherin-11 orcadherin-11 counter-receptor antisense molecules which inhibit thetranscription or translation of the foregoing nucleic acid molecules.

In one embodiment, a cadherin-11 modulating nucleic acid such as, forexample, a cadherin-11 inhibitory nucleic acid, (1) hybridizes understringent conditions to a nucleic acid having a sequence of SEQ ID NO:1, and (2) codes for a cadherin-11 polypeptide or a fragment (preferablyunique) thereof that is capable of binding specifically to cadherin-11or a cadherin-11 counter-receptor. In one embodiment, the cadherin-11polypeptide, or fragment thereof, binds to a cadherin-11counter-receptor on the surface of another cell, and thereby, inhibitsthe binding of cadherin-11 to its counter-receptor and, optionallyinhibits the binding of one cell to another. The inhibition of thebinding of one cell to the extracellular matrix is also envisioned. Thepreferred cadherin-11 inhibitory nucleic acid molecule has a nucleotidesequence of SEQ ID NO:1. The cadherin-11 modulating nucleic acidmolecules of the invention also include homologs and alleles of anucleic acid molecule having a sequence of SEQ ID NO:1.

The cadherin-11 modulating nucleic acid molecules, and preferably thecadherin-11 inhibitory nucleic acid molecules, may encode polypeptideswhich are soluble cadherin-11 polypeptides or membrane-boundpolypeptides. The soluble cadherin-11 polypeptides lack a transmembranedomain and, optimally, contain further amino acids which render thepolypeptide soluble (e.g., fusion proteins, containing all or part ofcadherin-11, which inhibit the binding of cadherin-11 to itscounter-receptor). Cadherin-11 modulating agents which aremembrane-bound (or membrane associated) preferably contain atransmembrane domain.

Cadherin-11 modulating nucleic acid molecules, and preferablycadherin-11 inhibitory nucleic acid molecules, further embrace nucleicacid molecules which code for a cadherin-11 polypeptide having the aminoacid sequence of SEQ ID NO: 2, but which may differ from the sequence ofSEQ ID NO: 1 due to the degeneracy of the genetic code. Similarly, thecadherin-11 modulating nucleic acid molecule, and preferably thecadherin-11 inhibitory nucleic acid molecule, may encode cadherin-11counter-receptor polypeptides and fragments thereof which are soluble ormembrane-bound.

In some important embodiments, the nucleic acids which modulate cellularfunction of cadherin-11 preferably do not encode a cadherin-11 or acadherin-11 counter-receptor polypeptide or a fragment thereof that iscapable of inhibiting binding of cadherin-11 to a cadherin-11counter-receptor, nor are they antisense molecules that inhibit thetranscription or translation of the foregoing nucleic acid molecules.

The cadherin-11 modulating nucleic acid molecules of the invention canbe identified by conventional techniques, e.g., by identifying nucleicacid sequences which code for cadherin-11 polypeptide and whichhybridize to a nucleic acid molecule having the sequence of SEQ ID NO: 1under stringent conditions. The term “stringent conditions”, as usedherein, refers to parameters with which the art is familiar. Morespecifically, stringent conditions, as used herein, refer tohybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%formamide, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin, 2.5mM NaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodiumchloride/0.15 M sodium citrate, pH 7; SDS is sodium dodecyl sulphate;and EDTA is ethylenediaminetetraacetic acid. After hybridization, themembrane to which the DNA is transferred is washed at 2×SSC at roomtemperature and then at 0.1×SSC/0.1×SDS at 65° C.

There are other conditions, reagents, and so forth which can be used,which result in a similar degree of stringency. The skilled artisan willbe familiar with such conditions and, thus, they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs. and alleles of the nucleic acid molecules of the invention.The skilled artisan also is familiar with the methodology for screeningcells and libraries for the expression of molecules, such as cadherin-11inhibitory nucleic acid molecules, which can be isolated and sequenced.In screening for cadherin-11 sequences for example, a Southern blot maybe performed using the foregoing conditions, together with a radioactiveprobe. After washing the membrane to which the DNA is finallytransferred, the membrane can be placed against x-ray film to detect theradioactive signal.

In general, cadherin-11 homologs and alleles typically will share atleast 70% nucleotide identity with SEQ. ID. NO: 1; and in someinstances, will share at least 75% nucleotide identity; and in stillother instances, will share at least 80% nucleotide identity.Watson-Crick complements of the foregoing nucleic acids are alsoembraced by the invention. The preferred cadherin-11 homologs have atleast 85% sequence homology to SEQ. ID. NO: 1. More preferably thecadherin-11 homologs have at least 90% and most preferably at least 95%sequence homology to SEQ. ID. NO: 1. The homology can be calculatedusing various, publicly available software tools developed by NCBI(Bethesda, Md.) that can be obtained through the internet at the NCBIwebsite. Exemplary tools include the BLAST system available at the NCBIwebsite. Pairwise and ClustalW alignments (BLOSUM30 matrix setting) aswell as Kyte-Doolittle hydropathic analysis can be obtained using theMacVetor sequence analysis software (Oxford Molecular Group).

The invention also includes degenerate nucleic acids which includealternative codons to those present in the naturally occurring nucleicacid that encodes, for example, the human cadherin-11 polypeptide. As iswell known in the art, and as an example, serine residues are encoded bythe codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons isequivalent for the purposes of encoding a serine residue. Thus, it willbe apparent to one of ordinary skill in the art that any of theserine-encoding nucleotide codons may be employed to direct the proteinsynthesis apparatus, in vitro or in vivo, to incorporate a serineresidue. Similarly, nucleotide sequence triplets which encode otheramino acid residues include, but are not limited to, CCA, CCC, CCG andCCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons);ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparaginecodons); and ATA, ATC and ATT (isoleucine codons). Other amino acidresidues may be encoded similarly by multiple nucleotide sequences.

It should be understood that homologs and alleles and degenerates ofnucleic acid molecules coding for a cadherin-11 counter-receptor arealso embraced by the invention and can be identified using the sametypes of techniques and procedures described herein in reference to thecadherin-11 homologs and alleles and degenerates of nucleic acidmolecules coding for a cadherin-11.

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

The nucleic acid molecule of the invention (e.g., cadherin-11 inhibitorynucleic acid molecule), in one embodiment, is operably linked to a geneexpression sequence which directs the expression of the cadherin-11inhibitory nucleic acid molecule within a eukaryotic cell. The “geneexpression sequence” is any regulatory nucleotide sequence, such as apromoter sequence or promoter-enhancer combination which facilitates theefficient transcription and translation of the cadherin-11 inhibitorynucleic acid molecule to which it is operably linked. The geneexpression sequence may, for example, be a mammalian or viral promoter,such as a constitutive or inducible promoter. Constitutive mammalianpromoters include, but are not limited to, the promoters for thefollowing genes: hypoxanthine phosphoribosyl transferase (HPTR),adenosine deaminase, pyruvate kinase, β-actin promoter and otherconstitutive promoters. Exemplary viral promoters which functionconstitutively in eukaryotic cells include, for example, promoters fromthe simian virus, papilloma virus, adenovirus, human immunodeficiencyvirus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminalrepeats (LTR) of moloney leukemia virus and other retroviruses, and thethymidine kinase promoter of herpes simplex virus. Other constitutivepromoters are known to those of ordinary skill in the art. The promotersuseful as gene expression sequences of the invention also includeinducible promoters. Inducible promoters are expressed in the presenceof an inducing agent. For example, the metallothionein promoter isinduced to promote transcription and translation in the presence ofcertain metal ions. Other inducible promoters are known to those ofordinary skill in the art.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined cadherin-11 inhibitory nucleic acid molecule. The gene expressionsequences optionally includes enhancer sequences or upstream activatorsequences as desired.

The invention intends to embrace nucleic acid molecules which code forcadherin-11 polypeptides and fragments thereof, cadherin-11counter-receptor polypeptides and fragments thereof, and cadherin-11inhibitory agents as described herein. These nucleic acid molecules maybe used in both in vivo treatment methods or in vitro screening assays.When expression of the nucleic acid is preferred (as opposed to someembodiments of antisense treatment methods), the nucleic acid moleculeis linked to a gene expression sequence which permits expression of thenucleic acid molecule. Nucleic acid molecules of the invention may beintroduced into a cell in vitro, followed by the transfer of the cell tothe site of inflammation. The cell into which the nucleic acid moleculeis introduced may be harvested from the site of inflammation (e.g., alymphocyte, such as a T cell, a synovioctye, such as a type Bsynovioctye, a mast cell, a macrophage, a dendritic cell, a plasma cell,a synovial-derived fibroblast, a synovial membrane lining cell, anosteoblast, a cartilage-derived cell or an invasive pannus-derived cell)or it may be a cell which is not normally present at the site ofinflammation. A sequence which permits expression of the nucleic acid ina cell located in a joint, such as for example, a synoviocyte, is onewhich is selectively transcriptionally active in the cell and therebycauses the expression of the nucleic acid in such a cell. Those ofordinary skill in the art will be able to easily identify alternativepromoters that are capable of expressing such a nucleic acid molecule ina synoviocyte, an osteoblast, a cartilage-derived cell, a lymphocyte orany other cell type mentioned herein. Alternatively, the transduced cellmay be cultured in vitro in order to produce a cadherin-11 modulatingagent (e.g., cadherin-11 inhibitory agent) or it may be used in in vitroscreening assays. For example, the gene expression sequence may be usedto express a cadherin-11 nucleic acid molecule in a cell which does notinherently express cadherin-11. The cadherin-11 nucleic acid moleculemay also be expressed in a cell which inherently expresses neithercadherin-11 nor cadherin-11 counter-receptor.

The nucleic acid molecule sequences of the invention (e.g., cadherin-11inhibitory nucleic acid molecules) and the gene expression sequence aresaid to be “operably linked” when they are covalently linked in such away as to place the transcription and/or translation. of the nucleicacid molecule sequence (e.g., a cadherin-11 coding sequence) under theinfluence or control of the gene expression sequence. If it is desiredthat nucleic acid molecule be translated into a functional protein, twoDNA sequences are said to be operably linked if induction of a promoterin the 5′ gene expression sequence results in the transcription of thenucleic acid molecule and if the nature of the linkage between the twoDNA sequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the nucleic acid molecule, or (3) interferewith the ability of the corresponding RNA transcript to be translatedinto a polypeptide. Thus, a gene expression sequence would be operablylinked to a nucleic acid molecule if the gene expression sequence werecapable of effecting transcription of that nucleic acid molecule suchthat the resulting transcript might be translated into the desiredpolypeptide.

In still other embodiments, the cadherin-11 modulating agent is anucleic acid molecule that, rather than encoding a polypeptide,functions as an antisense molecule. Antisense oligonucleotides thatselectively bind to a nucleic acid molecule encoding a cadherin-11polypeptide, or a fragment thereof, or a cadherin-11 counter-receptor(e.g., an integrin or one or more of its subunits), or a fragmentthereof, to decrease cadherin-11 mediated adhesion are embraced by thepresent invention. When using antisense preparations of the invention,local administration to the synovium or synovial fluid is preferred.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon SEQ ID NO:1 or upon allelic or homologous genomicand/or cDNA sequences, one of skill in the art can easily choose andsynthesize any of a number of appropriate antisense molecules for use inaccordance with the present invention. In order to be sufficientlyselective and potent for inhibition, such antisense oligonucleotidesshould comprise at least 10 and, more preferably, at least 15consecutive bases which are complementary to the target, although incertain cases modified oligonucleotides as short as 7 bases in lengthhave been used successfully as antisense oligonucleotides (Wagner etal., Nat. Med. 1(11):1 116-118, 1995). Most preferably, the antisenseoligonucleotides comprise a complementary sequence of 20-30 bases.

Although oligonucleotides may be chosen which are antisense to anyregion of the gene or mRNA transcripts, in preferred embodiments theantisense oligonucleotides correspond to N-terminal or 5′ upstream sitessuch as translation initiation, transcription initiation or promotersites. In addition, 3′-untranslated regions may be targeted by antisenseoligonucleotides. Targeting to mRNA splicing sites has also been used inthe art but may be less preferred if alternative mRNA splicing occurs.In addition, the antisense is targeted, preferably, to sites in whichmRNA secondary structure is not expected (see, e.g., Sainio et al., CellMol. Neurobiol. 14(5):439-457, 1994) and at which proteins are notexpected to bind. Finally, although SEQ ID NO:1 discloses a cDNAsequence, one of ordinary skill in the art may easily derive the genomicDNA corresponding to this sequence. Thus, the present invention alsoprovides for antisense oligonucleotides which are complementary to thegenomic DNA corresponding to SEQ ID NO:1. Similarly, antisense toallelic or homologous cadherin-11 or alternatively, cadherin-11counter-receptor cDNAs and genomic DNAs are enabled without undueexperimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose.

The present invention, thus, contemplates pharmaceutical preparationscontaining modified antisense molecules that are complementary to andhybridizable with, under physiological conditions, nucleic acidsencoding cadherin-11 or cadherin-11 counter-receptor polypeptides,together with pharmaceutically acceptable carriers. Antisenseoligonucleotides may be administered as part of a pharmaceuticalcomposition. Such a pharmaceutical composition may include the antisenseoligonucleotides in combination with any standard physiologically and/orpharmaceutically acceptable carriers which are known in the art. Thecompositions should be sterile and contain a therapeutically effectiveamount of the antisense oligonucleotides in a unit of weight or volumesuitable for administration to a patient.

The nucleic acids of the invention can be delivered to, for example, thesynovium and to cells of the joint alone or in association with avector. In its broadest sense, a “vector” is any vehicle capable offacilitating: (1) delivery of a nucleic acid molecule to a target celland/or (2) uptake of a nucleic acid molecule by a target cell.Preferably, the vectors transport the cadherin-11 modulating nucleicacid molecule into the target cell with reduced degradation relative tothe extent of degradation that would result in the absence of thevector. Optionally, a “targeting ligand” can be attached to the vectorto selectively deliver the vector to a cell which expresses on itssurface the cognate receptor for the targeting ligand. In this manner,the vector (containing, for example, a cadherin-11 inhibitory nucleicacid molecule) can be selectively delivered to the synovial lining in ajoint capsule. Methodologies for targeting include conjugates, such asthose described in U.S. Pat. No. 5,391,723. Preferably, the nucleic acidmolecules of the invention are targeted for delivery to a cell locatedin a joint capsule, such as a synoviocyte or a lymphocyte.

In general, the vectors useful in the invention are divided into twoclasses: biological vectors and chemical/physical vectors. Biologicalvectors are useful for delivery/uptake of nucleic acids to/by a targetcell. Biological vectors include, but are not limited to, plasmids,phagemids, viruses, other vehicles derived from viral or bacterialsources that have been manipulated by the insertion or incorporation ofthe nucleic acid sequences of the invention, and additional nucleic acidfragments (e.g., enhancers, promoters) which can be attached to thenucleic acid sequences of the invention. Viral vectors are a preferredtype of biological vector and include, but are not limited to, nucleicacid sequences from the following viruses: adenovirus; adeno-associatedvirus; retrovirus, such as moloney murine leukemia virus; harvey murinesarcoma virus; murine mammary tumor virus; rouse sarcoma virus;SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papillomaviruses; herpes virus; vaccinia virus; polio virus; and RNA virus suchas a retrovirus. One can readily employ other vectors not named butknown in the art.

A particularly preferred virus for certain applications is theadeno-associated virus, a double-stranded DNA virus. Theadeno-associated virus is capable of infecting a wide range of celltypes and species and can be engineered to be replication-deficient. Itfurther has advantages, such as heat and lipid solvent stability, hightransduction frequencies in cells of diverse lineages, includinghemopoietic cells, and lack of superinfection inhibition thus allowingmultiple series of transductions. Reportedly, the adeno-associated viruscan integrate into human cellular DNA in a site-specific manner, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

In general, other preferred viral vectors are based on non-cytopathiceukaryotic viruses in which non-essential genes have been replaced withthe gene of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Adenoviruses and retroviruses have been approved for human gene therapytrials. In general, the retroviruses are replication-deficient (i.e.,capable of directing synthesis of the desired proteins, but incapable ofmanufacturing an infectious particle). Such genetically alteredretroviral expression vectors have general utility for thehigh-efficiency transduction of genes in vivo. Standard protocols forproducing replication-deficient retroviruses (including the steps ofincorporation of exogenous genetic material into a plasmid, transfectionof a packaging cell line with plasmid, production of recombinantretroviruses by the packaging cell line, collection of viral particlesfrom tissue culture media, and infection of the target cells with viralparticles) are provided in Kriegler, M., “Gene Transfer and Expression,A Laboratory Manual,” W.H. Freeman C.O., New York (1990) and Murry, E.J. Ed. “Methods in Molecular Biology,” vol. 7, Humana Press, Inc.,Cliffton, N.J. (1991). Another preferred retroviral vector is the vectorderived from the moloney murine leukemia virus, as described in Nabel,E. G., et al., Science, v. 249, p. 1285-1288 (1990).

In addition to the biological vectors, chemical/physical vectors areuseful for delivery/uptake of nucleic acids or polypeptides to/by atarget cell. As used herein, a “chemical/physical vector” refers to anatural or synthetic molecule, other than those derived frombacteriological or viral sources, capable of delivering the cadherin-11modulating agent to a cell.

A preferred chemical/physical vector of the invention is a colloidaldispersion system. Colloidal dispersion systems include lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vessels which are useful as a deliveryvector in vivo or in vitro. It has been shown that large unilamellarvessels (LUV), which range in size from 0.2-4.0 μM can encapsulate largemacromolecules. RNA, DNA, and intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al., Trends Biochem. Sci., v. 6, p. 77 (1981)). Inorder for a liposome to be an efficient gene transfer vector, one ormore of the following characteristics should be present: (1)encapsulation of the gene of interest at high efficiency with retentionof biological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation.

Liposomes may be targeted to a particular tissue, by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein specific for the particular tissue or cell type(e.g., synoviocyte-specific surface proteins). Additionally, the vectormay be coupled to a nuclear targeting peptide, which will direct thecadherin-11 modulating nucleic acid molecule to the nucleus of the hostcell.

Liposomes are commercially available from Gibco BRL, for example, asLIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids suchas N-[1-(2,3 dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications. Liposomes also have been reviewed by Gregoriadis, G.in Trends in Biotechnology, V. 3, p. 235-241 (1985).

In general, the cadherin-11 modulating nucleic acid molecules can beadministered to the subject (any mammalian recipient) using the samemodes of administration that currently are used for gene therapy inhumans (e.g., adenovirus-mediated gene therapy). A patented procedurefor performing ex vivo gene therapy is outlined in U.S. Pat. No.5,399,346 and in exhibits submitted in the file history of that patent,all of which are publicly available documents. In general, ex vivo genetherapy involves introduction in vitro of a functional copy of a gene orfragment thereof into a cell(s) of a subject and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene or fragment thereof is under operable control of regulatoryelements which permit expression of the gene in the geneticallyengineered cell(s). Accordingly, the nucleic acids of the invention,including the cadherin-11 inhibitory nucleic acid molecules, can bedelivered to synoviocytes or lymphocytes or other cells of the jointcapsule ex vivo or in vivo, to treat an inflammatory joint disorder.Numerous transfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654.

As an illustrative example, a vector containing a nucleic acid moleculeis delivered to a site of joint inflammation in a subject who is acandidate for such gene therapy. Then, the vector genetically modifiesthe synoviocytes, osteoblasts, hemopoietic cells such as macrophages andNK cells, and/or lymphocytes in vivo with DNA encoding, for example, acadherin-11 inhibitory polypeptide of the invention. Such geneticallymodified joint cells are expected to interfere with cadherin-11 bindingto its counter-receptor in vivo.

In an alternative embodiment, primary human synoviocytes can be obtainedfrom a subject who is a candidate for such gene therapy. Then, suchcells can be genetically engineered ex vivo with DNA encoding, forexample, a cadherin-11 inhibitory polypeptide of the invention. Suchrecombinant cells are expected to inhibit cadherin-11 mediated adhesionin vivo. In yet another example, another cell type which expressesneither cadherin-11 nor cadherin-11 counter-receptor can be geneticallymanipulated in vitro to express a cadherin-11 inhibitory agent and thenintroduced into the site of inflammation.

Exemplary compositions that can be used to facilitate in vitro uptake ofthe nucleic acid molecules by a target cell include calcium phosphateand other chemical mediators of intracellular transport, microinjectioncompositions, electroporation and homologous recombination compositions(e.g., for integrating a nucleic acid into a preselected location withinthe target cell chromosome).

The invention also provides isolated unique fragments of an isolatednucleic acid molecule selected from the group consisting of SEQ ID NO:1.A unique nucleic acid fragment is one that is a ‘signature’ for thelarger nucleic acid. For example, the unique fragment is long enough toassure that its precise sequence is not found in molecules within thehuman genome outside of the cadherin-11 nucleic acid molecules definedherein. Those of ordinary skill in the art may apply no more thanroutine procedures to determine if a fragment is unique within the humangenome.

The invention further provides a pharmaceutical composition (i.e., apharmaceutical preparation) for modulating a cadherin-11 mediatedfunction in a subject. The composition includes a pharmaceuticallyacceptable carrier and a cadherin-11 modulating agent (e.g., acadherin-11 inhibitory agent).

The pharmaceutical preparations, as described above, are administered ineffective amounts. For therapeutic applications, it is generally thatamount sufficient to achieve a medically desirable result. In general, atherapeutically effective amount is that amount necessary to delay theonset of, inhibit the progression of, or halt altogether the particularcondition being treated. As an example, the effective amount isgenerally that amount which serves to alleviate the symptoms (e.g.,pain, inflammation, etc.) of the disorders described herein. Theeffective amount will depend upon the mode of administration, theparticular condition being treated and the desired outcome. It will alsodepend upon the stage of the condition, the severity of the condition,the age and physical condition of the subject being treated, the natureof concurrent therapy, if any, the duration of the treatment, thespecific route of administration and like factors within the knowledgeand expertise of the medical practitioner. For prophylacticapplications, it is that amount sufficient to delay the onset of,inhibit the progression of, or halt altogether the particular conditionbeing prevented, and may be measured by the amount required to preventthe onset of symptoms.

Generally, doses of active compounds of the present invention would befrom about 0.01 mg/kg per day to 1000 mg/kg per day, preferably fromabout 0.1 mg/kg to 200 mg/kg and most preferably from about 0.2 mg/kg toabout 20 mg/kg, in one or more dose administrations daily, for one ormore days. It is expected that doses ranging from 1-500 mg/kg, andpreferably doses ranging from 1-100 mg/kg, and even more preferablydoses ranging from 1-50 mg/kg, will be suitable. The preferred amountcan be determined by one of ordinary skill in the art in accordance withstandard practice for determining optimum dosage levels of the agent. Itis generally preferred that a maximum dose of a cadherin-11 modulatingagent that is the highest safe dose according to sound medical judgementbe used.

The cadherin-11 modulating agents of the invention can be administeredto a subject in need of such treatment in combination with concurrenttherapy for treating an inflammatory joint disorder. The concurrenttherapy may be invasive, such as a removal of fluid from the jointcapsule, or may involve drug therapy such as the administration ofnonsteroidal anti-inflammatory drugs, anti-rheumatic drugs (e.g., goldand pencillamine), immune modulating drugs (e.g., cyclosporin) andcorticosteroid drugs. These drug therapies are well-known to those ofordinary skill in the art and are administered by modes known to thoseof such skill. The drug therapies are administered in amounts which areeffective to achieve the physiological goals (e.g., to reduceinflammation in a joint), in combination with, for example, thecadherin-11 inhibitory agent of the invention. Thus, it is contemplatedthat the drug therapies may be administered in amounts which are notcapable of preventing or reducing the physiological consequences of aninflammatory joint disorder when the drug therapies are administeredalone but which are capable of reducing the consequences whenadministered in combination with the cadherin-11 modulating agents ofthe invention.

The cadherin-11 modulating agent may be administered alone or incombination with the above-described drug therapies as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the cadherin-11 modulating agent in combination with anystandard physiologically and/or pharmaceutically acceptable carrierswhich are known in the art. The compositions should be sterile andcontain a therapeutically effective amount of the cadherin-11 modulatingagent in a unit of weight or volume suitable for administration to apatient.

The term “pharmaceutically-acceptable carrier” as used herein means oneor more compatible solid or liquid filler, diluents or encapsulatingsubstances which are suitable for administration into a human or otheranimal. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients. Pharmaceuticallyacceptable further means a non-toxic material that is compatible with abiological system such as a cell, cell culture, tissue, or organism. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The characteristics of the carrier will depend on theroute of administration. The components of the pharmaceuticalcompositions also are capable of being commingled with the agents of thepresent invention, and with each other, in a manner such that there isno interaction which would substantially impair the desiredpharmaceutical efficacy. The pharmaceutically acceptable carrier must besterile for in vivo administration. Physiologically and pharmaceuticallyacceptable carriers include diluents, fillers, salts, buffers,stabilizers, solubilizers, and other materials which are well known inthe art.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the cadherin-11 modulatingagents, which is preferably isotonic with the blood of the recipient.This aqueous preparation may be formulated according to known methodsusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation also may be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may beused in the preparation of injectables. Carrier formulations suitablefor oral, subcutaneous, intravenous, intramuscular, etc. administrationscan be found in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular drug selected, theseverity of the condition being treated, and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, interdermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Intravenous orintramuscular routes are not particularly suitable for long-term therapyand prophylaxis. They could, however, be preferred in emergencysituations. Oral administration will be preferred for prophylactictreatment because of the convenience to the patient as well as thedosing schedule. In preferred embodiments, the pharmaceuticalcomposition is administered directly to the synovium, synovial fluid orjoint capsule by injection preferably with a syringe.

Formulations for use in accordance with the methods of the inventioninclude a syringe containing a cadherin-11 modulating agent, such as acadherin-11 inhibitory agent, and a pharmaceutically acceptable carrierthat is suitable for injection into the synovial fluid.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing thecadherin-11 modulating agents into association with a carrier whichconstitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing thecadherin-11 modulating agents into association with a liquid carrier, afinely divided solid carrier, or both, and then, if necessary, shapingthe product. Compositions suitable for oral administration may bepresented as discrete units, such as capsules, tablets, lozenges, eachcontaining a predetermined amount of the cadherin-11 modulating agent.Other compositions include suspensions in aqueous liquids or non-aqueousliquids such as a syrup, elixir or an emulsion.

In one particular embodiment, the preferred vehicle for delivery of thecadherin-11 modulating agents of the invention is a biocompatiblemicroparticle or implant that is suitable for implantation into a jointor in the vicinity of an afflicted joint in the recipient. Exemplarybioerodible implants that are useful in accordance with this method aredescribed in PCT International application no. PCT/US/03307 (PublicationNo. WO 95/24929, entitled “Polymeric Gene Delivery System”, claimingpriority to U.S. patent application Ser. No. 213,668, filed Mar. 15,1994). PCT/US/0307 describes a biocompatible, preferably biodegradablepolymeric matrix for containing an exogenous gene under the control ofan appropriate promoter. The polymeric matrix is used to achievesustained release of the exogenous gene in the subject. In accordancewith the instant invention, the cadherin-11 modulating agents describedherein are encapsulated or dispersed within the biocompatible,preferably biodegradable polymeric matrix disclosed in PCT/US/03307. Thepolymeric matrix preferably is in the form of a microparticle such as amicrosphere (wherein, for example, the cadherin-11 inhibitory agent isdispersed throughout a solid polymeric matrix) or a microcapsule(wherein, for example, the cadherin-11 inhibitory agent is stored in thecore of a polymeric shell). Other forms of the polymeric matrix forcontaining the cadherin-11 modulating agent include films, coatings,gels, implants, and stents. The size and composition of the polymericmatrix device is selected to result in favorable release kinetics in thetissue into which the matrix device is implanted. The size of thepolymeric matrix devise is further selected according to the method ofdelivery which is to be used, typically injection into a tissue, such asthe joint, joint capsule, synovial membrane or synovial fluid, oradministration of a suspension by aerosol into the nasal and/orpulmonary areas. The polymeric matrix composition can be selected tohave both favorable degradation rates and also to be formed of amaterial which is bioadhesive, to further increase the effectiveness oftransfer when the devise is administered to the synovium of a joint. Thematrix composition also can be selected not to degrade, but rather, torelease by diffusion over an extended period of time.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the cadherin-11 modulating agents, including the cadherin-11inhibitory agents of the invention to the subject. Biodegradablematrices are preferred. Such polymers may be natural or syntheticpolymers. Synthetic polymers are preferred. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multi-valent ions or other polymers.

In general, the cadherin-11 modulating agents of the invention aredelivered using the bioerodible implant by way of diffusion, or morepreferably, by degradation of the polymeric matrix. Exemplary syntheticpolymers which can be used to form the biodegradable delivery systeminclude: polyamides, polycarbonates, polyalkylenes, polyalkyleneglycols, polyalkylene oxides, polyalkylene terepthalates, polyvinylalcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene and polyvinylpyrrolidone.

Examples of biodegradable polymers include synthetic polymers such aspolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),and poly(lactide-co-caprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodiblehydrogels (described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein), polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the cadherin-11 modulating agents described above,increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available and known to those of ordinaryskill in the art. They include the above-described polymeric systems, aswell as polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono- di- andtri-glycerides; hydrogel release systems; silastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe cadherin-11 modulating agent is contained in a form within a matrixsuch as those described in U.S. Pat. Nos. 4,452,775, 4,675,189 and5,736,152 and (b) diffusional systems in which an active. componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-basedhardware delivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

Another aspect of the invention includes a screening assay method fordetermining whether a putative cadherin-11 inhibitory agent modulatescadherin-11 mediated adhesion. In certain embodiments, an in vitroadhesion assay is used as a screening assay to measure the ability of anagent, e.g., a pharmaceutical lead compound, an antibody or a fragmentthereof, a library member or a peptide analog, to inhibitcadherin-1′-mediated adhesion between a first cell expressingcadherin-11 and a second cell expressing a cadherin-11 counter-receptorin vitro. The assay is predictive of the ability of the agent to inhibitcadherin-11 mediated activity in vivo.

The binding partners in the adhesion assays are the particular ligandsand receptors involved in cadherin-11 mediated adhesion. Accordingly,adhesion assays can be performed in which the binding partners are: (1)a cell expressing a cadherin such as cadherin-11 (e.g., synoviocytes,synovial-derived fibroblasts, osteoblasts and cadherin-11 transfectedcells) and a cell expressing a cadherin-11 counter-receptor (e.g.,synoviocytes, synovial-derived fibroblasts, osteoblasts, T and Blymphocytes, plasma cells, macrophages, dendritic cells, mast cells, NKcells and cadherin-11 counter-receptor transfected cells; (2) anisolated cadherin-11 and a cell expressing the counter-receptor (3) anisolated cadherin-11 counter-receptor and a cell expressing cadherin-11and; (4) an isolated cadherin-11 polypeptide and its isolatedcounter-receptor. When isolated cadherin-11 or an isolated cadherin-11counter-receptor are used, these may be present in immobilized form(e.g., immobilized on a solid surface) or in soluble form.

As used herein with respect to adhesion assays, a cadherin-11counter-receptor can be present as an isolated cadherin-11counter-receptor, a functionally equivalent peptide fragment or analogof the isolated cadherin-11 counter-receptor, or a cell expressing thecounter-receptor extracellularly or its functionally equivalent peptidefragment or analog. Similarly, cadherin-11 can be present as an isolatedcadherin-11 polypeptide, a functionally equivalent peptide fragment oranalog of cadherin-11, or a cell expressing cadherin-11 extracellularlyor its functionally equivalent peptide fragment or analog. Cadherin-11or its counter-receptor can be immobilized on supports, such asmicrotiter plates or beads, using procedures known to the artisan ofordinary skill in the art.

Thus, a high throughput screening assay for selecting pharmaceuticallead compounds can be performed in which, for example, (1) cadherin-11or the cadherin-11 counter-receptor is immobilized onto the surface of amicrotiter well, (2) aliquots of a molecular library containing librarymembers are added to the wells, (3) cells expressing a cadherin-11counter-receptor or cadherin-11 (as the case may be) are added to thewells and (4) the well components are allowed to incubate for a periodof time that is sufficient for the cells to bind to the immobilizedcadherin-11. Preferably, the cells are labeled (e.g., preincubated with⁵¹Cr or a fluorescent dye) prior to their addition to the microtiterwell. Following the incubation period, the wells are washed to removenon-adherent cells and the signal (attributable to the label on theadhering cells) is determined. A positive control (e.g., no librarymember present) on the same microtiter plate is used to establishmaximal adhesion value. A negative control (e.g., soluble cadherin-11added to the microtiter well) on the same microtiter plate is used toestablish maximal levels of inhibition of adhesion. As an example,cadherin-11 can be immobilized to the surface and the cells added can beT cells. Consistent with other embodiments of the invention, the highthroughput screening assay may also use an isolated cadherin-11 and/orcadherin-11 counter-receptor, both of which may be in immobilized orsoluble form.

The agents to be screened may be pharmaceutical lead compoundssynthesized in molecular libraries. These libraries can yield peptides(i.e., peptide libraries) or small organic or inorganic molecules. Theagents to be screened can also be peptide analogs of either cadherin-11or cadherin-11 counter-receptor. Preferably, the peptide or peptideanalog of cadherin-11 or cadherin-11 counter-receptor corresponds to theportion of either polypeptide responsible for binding with its bindingpartner. For both polypeptides, this portion is extracellular.

According to yet another aspect of the invention, a method is providedfor screening a molecular library to identify pharmaceutical leadmolecules which inhibit the in vitro adhesion between a first cell thatexpresses cadherin-11 and a second cell that expresses a cadherin-11counter receptor. For example, the ability of a molecule to inhibit thebinding of a synovial cell to a T-lymphocyte or the binding of asynovial cell to another synovial cell in vitro can be used as ascreening assay to identify lead compounds which inhibit the binding ofcadherin-11 to its counter-receptor. Such adhesion assays are well knownin the art and are illustrated by the assay provided in the Examples.

A preferred screening method involves performing an adhesion assaybetween a first cell and a second cell in the presence and absence of atleast one member of the molecular library to determine whether thelibrary member modulates adhesion between the first cell and the secondcell in vitro. Preferably the first cell expresses cadherin-11 and thesecond cell expresses a cadherin-11 counter-receptor. This embodimentinvolves: (1) performing a first adhesion assay between the first celland the second cell to obtain a first adhesion assay result; (2)performing a second adhesion assay between the first cell and the secondcell in the presence of the library member to obtain a second adhesionassay result; and (3) comparing the first and the second adhesion assayresults to determine whether the library member modulates adhesionbetween the first cell and the second cell. A difference between thefirst and the second adhesion assay result indicates the ability of thelibrary member to modulate binding between the first cell and the secondcell and thus binding of cadherin-11 to its counter-receptor. Thus, forexample, an adhesion assay result which shows reduced binding betweenthe first cell and the second cell when the assay is conducted in thepresence of the library member, compared to the assay result obtainedwhen the assay is performed in the absence of the library member,indicates that the library member inhibits binding of the first cell andthe second cell. An exemplary adhesion assay is provided in theExamples. Other such adhesion assays are well known in the art and canbe developed and performed using no more than routine experimentation.Thus, for example, the adhesion assay can be performed by substitutingthe first cell and the second cell with an isolated cadherin-11 and itsisolated counter-receptor.

In yet another aspect, the invention provides a screening assay forpharmaceutical lead compounds which modulate cellular function throughcadherin-11. These screening assays involve determining a first valuefor a cellular parameter (e.g., cell proliferation) of a cadherin-11expressing cell (e.g., a synoviocyte) in the absence of a molecularlibrary member, determining a second value for the same cellularparameter in a cadherin-11 expressing cell in the presence of amolecular library member, and comparing the first and the second valueof the cellular parameter as a measure of the effect of the molecularlibrary member on that particular cellular parameter. As an example, asecond value which is lower than the first value is indicative of areduction in cellular proliferation. Cellular proliferation can bemeasured in any number of ways, well known to the ordinary artisan,including but not limited to incorporation of radioactive nucleotides(e.g., thymidine uptake assays) and counting of cells. An example of a Tcell proliferation assay is described in U.S. Pat. No. 6,077,833, whichis incorporated herein in its entirety by reference. In these assays,the cells to be used may be either adhered to a solid surface or may bepresent in a suspension.

Likewise, the assay can also be carried out by measuring other cellularparameters such as apoptosis, migration, attachment and/or factor (e.g.,inflammatory factor) secretion. For apoptosis screening assays, cellcounts may be used as a readout of the amount of apoptosis. Otherapoptosis assays are described in U.S. Pat. No. 6,107,088. For factorsecretion assays, the supernatant within which the cells exist can betested for the presence of factors, using other functional assays orimmunological assays (e.g., ELISA assays, bioassays, western blots, RIAassays, and so forth to detect particular cytokines). For example,secretion of IL-6 can be measured by using the supernatant in an assaymeasuring cell growth of IL-6 dependent cells. General immune cellactivation can also be measured using assays such as those described inU.S. Pat. No. 5,569,585.

In some preferred embodiments, the cellular function is a function otherthan cadherin-11 binding to a cadherin-11 counter-receptor. Thus, thepreferable pharmaceutical lead compound, according to this aspect of theinvention, is one which enhances or inhibits a cadherin-11 mediatedcellular function such as proliferation, signaling, apoptosis, factorproduction or secretion, migration or attachment, yet does not inhibitcadherin-11 binding to a cadherin-11 counter-receptor. Preferably, thecadherin-11 modulating agents which do not inhibit cadherin-11 bindingto its counter receptor, are selected from small molecule libraries,such as combinational chemistry libraries.

In all of the aforementioned assays, a preliminary binding screenoptionally is initially carried out prior to the screening assaysdescribed herein. Such a preliminary binding screen would enrich and insome instances identify pharmaceutical lead compounds which bindcadherin-11 or its counter-receptor. These preliminary assays would notnecessarily measure the inhibitory or signal modulating capability ofthe identified compounds, but rather would serve to reduce the number ofcompounds to be further screened. Thus, as an example, a molecularlibrary may be initially contacted with either (preferably immobilized)cadherin-11 or a cadherin-11 counter-receptor and the library memberswhich bind to cadherin-11 or cadherin-11 counter-receptor would befurther screened for their ability to inhibit cadherin-11 binding to itscounter-receptor or to modulate cellular function of cadherin-11expressing cells. Since agents which modulate cellular function do so incadherin-11 expressing cells, the appropriate preliminary screen forthese agents would be binding to cadherin-11. For inhibitory agents ofthe invention, the preliminary screen may test binding to eithercadherin-11 or cadherin-11 counter-receptor.

The cadherin-11 modulating agents of the invention can be synthesizedfrom peptides or other biomolecules including but not limited tosaccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines,derivatives or structural analogs of the above, or combinations thereofand the like. Phage display libraries and chemical combinatoriallibraries can be used to develop and select synthetic compounds whichare cadherin-11 modulating and/or inhibitory agents. Also envisioned inthe invention is the use of agents made from peptoids, randombio-oligomers (U.S. Pat. No. 5,650,489), benzodiazepines, diversomeressuch as dydantoins, benzodiazepines and dipeptides, nonpeptidalpeptidomimetics with a beta-D-glucose scaffolding, oligocarbamates orpeptidyl phosphonates.

The agents of the invention may be produced en masse using librarytechnology. In some aspects, the methods of the invention utilize thislibrary technology to generate and subsequently identify smallmolecules, including small peptides, which bind to a cadherin-11 or acadherin-11 counter receptor. One advantage of using libraries is thefacile manipulation of millions of different putative candidates ofsmall size in small reaction volumes (i.e., in synthesis and screeningreactions). Another advantage of libraries is the ability to synthesizeagents which might not otherwise be attainable using naturally occurringsources, particularly in the case of non-peptide moieties.

A “molecular library” refers to a collection of structurally-diversemolecules. Molecular libraries can be chemically-synthesized orrecombinantly produced. As used herein, a “molecular library member”refers to a molecule that is present within the molecular library. Ingeneral, a molecular library contains from two to 10¹² molecules, andany integer number therebetween, e.g., 2, 3, 4, 5, 10, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² and so forth, as if each andevery integer has been recited herein.

Methods for preparing libraries of molecules are well known in the artand many libraries are commercially available. Libraries of interest inthe invention include peptide libraries, randomized oligonucleotidelibraries, synthetic organic combinatorial libraries, and the like.Degenerate peptide libraries can be readily prepared in solution, inimmobilized form as bacterial flagella peptide display libraries or asphage display libraries. Peptide ligands can be selected fromcombinatorial libraries of peptides containing at least one amino acid.Libraries can be synthesized of peptoids and non-peptide syntheticmoieties. Such libraries can further be synthesized which containnon-peptide synthetic moieties which are less subject to enzymaticdegradation compared to their naturally-occurring counterparts.Libraries are also meant to include for example but are not limited topeptide on plasmid libraries, polysome libraries, aptamer libraries,synthetic peptide libraries, synthetic small molecule libraries andchemical libraries. The libraries can also comprise cyclic carbon orheterocyclic structure and/or aromatic or polyaromatic structuressubstituted with one or more of the above-identified functional groups.

Many of these agents of the invention may be synthesized usingrecombinant or chemical library approaches. A vast array of agents canbe generated from libraries of synthetic or natural compounds. Librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or can readily produced. Natural andsynthetically produced libraries and compounds can be readily modifiedthrough conventional chemical, physical, and biochemical means. Knownbinding partners of cadherin-11 may be subjected to directed or randomchemical modifications such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs of these bindingpartners, which may function as agonists or antagonists.

Libraries include recombinantly-produced libraries of fusion proteins.An exemplary recombinantly-produced library is prepared by ligatingfragments of cadherin-11 cDNA into, for example, the pGEX-2T vector(Pharmacia, Piscataway, N.J.). This vector contains the carboxy terminusof glutathione S-transferase (GST) from Schistosoma japonicum. Use ofthe GST-containing vector facilitates purification of GST-cadherin-11fusion proteins from bacterial lysates by affinity chromatography onglutathione sepharose. After elution from the affinity column,cadherin-11 GST fusion proteins are tested for activity by, for example,contacting at least one fusion protein with an cadherin-11 expressingcell prior to (or concurrently with) contacting the cadherin-11counter-receptor expressing cell with a cadherin-11 expressing cell.Fusion proteins which inhibit binding between the cadherin-11counter-receptor expressing cells and the cadherin-11 expressing cellsare selected as pharmaceutical lead compounds. These proteins are alsouseful in further characterization of the portion of cadherin-11 towhich the counter-receptor binds. See, for example, Koivunen E. et al.(1993) J. Biol. Chem. 268(27):20205 which describes the selection ofpeptides which bind to the α⁵β₁ integrin from a phage display library.

Synthetic DNA and RNA libraries are also commonly used in the art. Forinstance, Ellington and Szostak describe the use of randompolynucleotide libraries to identify novel ligands (Ellington andSzostak, Nature, 346, 818-822 (1990)). As an example, modificationswhich create variants of cadherin-11 can be made at the level of thenucleic acid sequence which encodes a cadherin-11 polypeptide. Aminoacid substitutions may be made by PCR-directed mutation, site-directedmutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad.Sci. U.S.A. 82: 488-492, 1985), or by chemical synthesis of the nucleicacid molecules encoding cadherin-11 or a cadherin-11 counter-receptor.

As described in U.S. Pat. No. 5,908,609, exemplary library compoundsalso include, but are not limited to, peptides such as, for example,soluble peptides, including but not limited to members of random peptidelibraries; (see, e.g., Lam, K. S. et al. 1991, Nature 354:82-84;Houghten, R. et al. 1991, Nature 354:84-86), and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids, phosphopeptides (including but not limited to, members ofrandom or partially degenerate, directed phosphopeptide libraries; (see,e.g., Songyang, Z. et al. 1993, Cell 72: 767-778), antibodies(including, but not limited to, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′)₂and FAb expression library fragments (and epitope-binding fragmentsthereof), and small organic or inorganic molecules. Other compoundswhich can be screened in accordance with the invention include but arenot limited to small organic molecules.

Compounds of the invention that may be designed to satisfy the foregoingcriteria include polypeptides and peptide mimetics. The peptide mimeticcan be a hybrid molecule which includes both amino acid and non-aminoacid components, e.g., the mimic can include amino acid components forthe positively charged and negatively charged regions and a non-aminoacid (e.g., piperidine) having the same approximate size and dimensionof a hydrophobic amino acid (e.g., phenylalanine) as the hydrophobiccomponent.

Small molecule combinatorial libraries may also be generated. Acombinatorial library of small organic compounds is a collection ofclosely related analogs that differ from each other in one or morepoints of diversity and are synthesized by organic techniques usingmulti-step processes. Combinatorial libraries include a vast number ofsmall organic compounds. One type of combinatorial library is preparedby means of parallel synthesis methods to produce a compound array. A“compound array” as used herein is a collection of compoundsidentifiable by their spatial addresses in Cartesian coordinates andarranged such that each compound has a common molecular core and one ormore variable structural diversity elements. The compounds in such acompound array are produced in parallel in separate reaction vessels,with each compound identified and tracked by its spatial address.Examples of parallel synthesis mixtures and parallel synthesis methodsare provided in U.S. Ser. No. 08/177,497, filed Jan. 5, 1994 and itscorresponding PCT published patent application W095/18972, publishedJul. 13, 1995 and U.S. Pat. No. 5,712,171 granted Jan. 27, 1998 and itscorresponding PCT published patent application W096/22529, which arehereby incorporated by reference.

Thus, the invention provides, in part, low molecular weight compoundsthat modulate cadherin-11 mediated functions. These compounds can beused to modulate cadherin-11 function or can be used as lead compoundsfor the design of better compounds using the computer-based rationaldrug design methods.

One of skill in the art will be familiar with methods for predicting theeffect on protein conformation of a change in protein sequence, and canthus “design” a variant which functions as modulating agent according toknown methods. One example of such a method is described by Dahiyat andMayo in Science 278:82-87, 1997, which describes the design of proteinsde novo. The method can be applied to a known protein to vary only aportion of the polypeptide sequence. By applying the computationalmethods of Dahiyat and Mayo, specific variants of cadherin-11 or acadherin-11 counter-receptor can be proposed and tested to determinewhether the variant retains a desired conformation. Similarly, Blake(U.S. Pat. No. 5,565,325) teaches the use of known ligand structures topredict and synthesize variants with similar or modified function.

Other methods for preparing or identifying peptides which bind to aparticular target are known in the art. Molecular imprinting, forinstance, may be used for the de novo construction of macromolecularstructures such as peptides which bind to a particular molecule. See,for example, Kenneth J. Shea, Molecular Imprinting of Synthetic NetworkPolymers: The De Novo synthesis of Macromolecular Binding and CatalyticSites, TRIP Vol. 2, No. 5, May 1994; Klaus Mosbach, MolecularImprinting, Trends in Biochem. Sci., 19(9) January 1994; and Wulff, G.,in Polymeric Reagents and Catalysts (Ford, W. T., Ed.) ACS SymposiumSeries No. 308, pp 186-230, American Chemical Society (1986). As anexample, one method for preparing mimics of cadherin-11counter-receptors involves the steps of: (i) polymerization offunctional monomers around a known substrate (the template or in thiscase, cadherin-11 counter-receptor binding domain) that exhibits adesired activity; (ii) removal of the template molecule; and then (iii)polymerization of a second class of monomers in the void left by thetemplate, to provide a new molecule which exhibits one or more desiredproperties which are similar to that of the template. In addition topreparing peptides in this manner other binding molecules such aspolysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins,carbohydrates, glycoproteins, steroids, lipids, and other biologicallyactive materials can also be prepared. This method is useful fordesigning a wide variety. of biological mimics that are more stable thantheir natural counterparts, because they are typically prepared by thefree radical polymerization of functional monomers, resulting in acompound with a nonbiodegradable backbone. Other methods for designingsuch molecules include for example drug design based on structureactivity relationships which require the synthesis and evaluation of anumber of compounds and molecular modeling.

As mentioned above, the invention further embraces agents comprisingpeptidomimetic residues, including non-naturally occurring amino acids.Such variants can be synthesized by substituting amino acids residuesinvolved in cadherin-11 mediated functions with peptidomimetic residues.For example, glutamine (Glu) residues may be replaced withα-aminoadipate molecules and tyrosine positions may be substituted with4-carboxymethyl-Phe. Phosphorus and non-phosphorus based analogs, suchas phosphorotyrosine mimetics, may be used in the variants. Tyrosineanalogs which can be used in place of the tyrosine residues includephenylalanine (Phe), pentafluoro phenylalanine (PfPhe),4-carboxymethyl-L-phenylalanine (cmPhe),4-carboxydifluoromethyl-L-phenylalanine (F₂ cmPhe),4-phosphonomethyl-phenylalanine (Pmp),(difluorophosphonomethyl)phenylalanine (F₂Pmp), O-malonyl-L-tyrosine(malTyr or OMT), and fluoro-O-malonyltyrosine (FOMT). Phosphonate-basedmimetics which substitute a methylene unit for the tyrosyl phosphateester bond may also be incorporated into synthetic agonists andantagonists. Additionally, glutamic acid residues can be modified topossess an additional methylene group or they may simply be substitutedwith α-amino-adipate (Adi). Other residues which may be used include thenon-naturally occurring amino acid 1-aminocyclohexylcarboxylic acid(Ac₆c) and 3-(2-hydroxynaphtalen-1-yl)-propyl, or 2-azetidinecarboxylicacid or pipecolic acid (which have 6-membered, and 4-membered ringstructures respectively) for proline residues, S-ethylisothiourea,2-NH₂-thiazoline and 2-NH₂-thiazole. Also useful in the synthesis ofvariants is the use of asparagine residue substitutes such as3-indolyl-propyl. It will be apparent to one of ordinary skill in theart that the invention embraces the synthesis of a wide variety ofvariants having any combination of amino acid analogs and/orpeptidomimetic residues as described above and as are known in the art.Further potential modifications envisioned by the invention includemodifications of cysteines, histidines, lysines, arginines, tyrosines,glutamines, asparagines, prolines, and carboxyl groups are well known inthe art and are described in U.S. Pat. No. 6,037,134. Synthesis of theafore-mentioned variants is described in the cited references and iswell within the realm of one of ordinary skill in the art.

The variants may also be modified to introduce or stabilize certainstructural features. As an example, β-bends may be incorporated intothe, preferably peptide, variants or the variants may be synthesized ascyclic peptides for example by incorporating thio-ether linkages.

The screening methods of the invention provide useful information forthe rational drug design of novel agents which are, for example, capableof modulating an immune system response. Exemplary procedures forrational drug design are provided in Saragovi, H. et al., (1992)Biotechnology 10:773; Haber E., (1983) Biochem. Pharmacol. 32(13):1967;and Connolly Y., (1991) Methods of Enzymology 203, Ch. 29“Computer-Assisted Rational Drug Design” pp 587-616, the contents ofwhich are incorporated herein by reference.

Thus, knowledge of the primary, secondary or tertiary structures ofnaturally occurring ligands and receptors can be used to rationallychoose or design molecules which will bind with either the ligand orreceptor. In particular, knowledge of the binding regions of ligands andreceptors can be used to rationally choose or design compounds which aremore potent than the naturally occurring ligands in eliciting theirnormal response or which are competitive inhibitors of theligand-receptor interaction.

Once rationally chosen or designed and selected, the library members maybe altered, e.g., in primary sequence, to produce new and differentpeptides. These fragments may be produced by site-directed mutagenesisor may be synthesized in vitro. These new fragments may then be testedfor their ability to bind to the receptor or ligand and, by varyingtheir primary sequences and observing the effects, peptides withincreased binding or inhibitory ability can be produced.

Alternatively, the nucleotide and amino acid sequences of thecadherin-11 modulating agents of the present invention, e.g., thosecorresponding to the extracellular domain of cadherin-11, may be used incomputer-based modeling systems to predict the secondary and tertiarystructure of the extracellular domain. Such computer-based systems arewell known to those of ordinary skill in the art of rational drugdesign. Based upon the tertiary structure of a receptor protein, it isoften possible to identify a binding region which is involved in itsbiological activity. From this information, peptides or other compoundswhich include or mimic this structure and/or which are capable ofbinding to it can be rationally designed. In this way, new compounds maybe designed which mimic the activity of the receptor or ligand or whichwill act as competitive inhibitors of the receptor or ligand.

It will be appreciated by those skilled in the art that variousmodifications of the foregoing peptide analogs can be made withoutdeparting from the essential nature of the invention. Accordingly, it isintended that peptides which include conservative substitutions andcoupled proteins in which a peptide of the invention is coupled to asolid support, such as a polymeric bead, a carrier molecule, such askeyhole limpet hemocyanin, or a reporter group, such as radiolabel orother tag, are also embraced within the teachings of the invention.

As used herein, “conservative amino acid substitution” refers to anamino acid substitution which does not alter the relative charge or sizecharacteristics of the peptide in which the amino acid substitution ismade. Conservative substitutions of amino acids include substitutionsmade amongst amino acids within the following groups: (a) MILV; (b) FYW;(c) KRH; (d) AG; (e) ST; (f) QN; and (g) ED.

The monoclonal antibodies of the invention which inhibit cadherin-11binding to its counter-receptor also are useful in screening assays foridentifying pharmaceutical lead compounds in molecular libraries. Theantibodies of the invention specifically bind to a cadherin or acadherin counter-receptor and thereby inhibit the binding of these twomolecules to each other. Thus, screening assays using monoclonalantibodies are also useful for assessing the ability of a librarymolecule to inhibit the binding of cadherin-11 to its counter-receptor.Such antibody-based screening assays are performed by contacting anantibody (that specifically binds to cadherin-11 and, for example,inhibits adhesion between a T lymphocyte and a cadherin-11 expressingcell) with cadherin-11 in the presence and absence of at least onemember of the molecular library and determining whether the librarymember modulates binding between the antibody and cadherin-11. In aparticularly preferred embodiment, cadherin-11 is presented as acadherin-11-expressing cell, an isolated cadherin-11 or an isolatedpeptide related to, or derived from, the extracellular domain ofcadherin-11. Similarly, the cadherin-11 counter-receptor can bepresented in any one of these fashions.

In a particularly preferred embodiment in which the antibody is ananti-cadherin-11 antibody, the antibody screening method involves: (1)performing a first antibody assay in the presence of the antibody andcadherin-11 and in the absence of the library molecule to obtain a firstantibody assay result; (2) performing a second antibody assay in thepresence of the antibody, cadherin-11 and the library molecule to obtaina second antibody assay result; and (3) comparing the first and thesecond antibody assay results to determine whether the molecular librarymember modulates binding between the antibody and cadherin-11. Accordingto this embodiment, reduced binding between the antibody and cadherin-11in the presence of the library member indicates that the library memberhas inhibited binding of antibody to cadherin-11. Antibody bindingassays also can be used to assess the relative ability of a molecularlibrary member to block binding between an antibody specific for thecadherin-11 counter-receptor and the counter-receptor using no more thanroutine experimentation.

These and other screening assays can also be used to identify thefunctionally equivalent cadherin-11 peptide analogs of the inventionthat are useful for inhibiting the binding of cadherin-11 to itscounter-receptor.

The cadherin-11 modulating agents can also be used, for example, totarget a toxin (e.g., ricin) or a detectable agent (e.g., a radiolabel,a fluorescent label, an enzyme label) to cells which express cadherin-11counter-receptors or cadherin-11. Methods for coupling such toxinsand/or agents to proteins and/or antibodies for in vivo and in vitroapplications are disclosed in, for example, Killen and Lindstrom (1984),“Specific killing of lymphocytes that cause experimental AutoimmuneMyestenia Gravis by toxin-acetylcholine receptor conjugates”, J. Immun.133:1335; Jansen, F. K., et al. (1982), “Immunotoxins: Hybrid moleculescombining high specificity and potent cytotoxicity”, Immunolog. Rev.62:185-216, the entire contents of which references are incorporatedherein by reference. See also U.S. Pat. Nos. 3,652,761; 4,478,946 and4,554,088, the entire contents of which patents are incorporated hereinby reference.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention. It is also to be understood thatthe reference figures are illustrative only and are not essential to theenablement of the claimed invention.

EXAMPLES

The importance of catenins in mediating cadherin dependent cell-to-celladhesion suggested that they might be used as probes to identify newcadherins. Thus, for these studies, we obtained antisera to both humanα-, and β-catenin and used them to identify co-precipitating proteins intype B human synoviocytes, as this cell type was not known to express acadherin. In addition, based upon alignments of the human E-, P-, andN-cadherin, four regions of identity can be appreciated. Using theseregions we synthesized sense and antisense oligonucleotides andperformed PCR under normal stringency conditions using RNA from humantype B synoviocytes as the template. The PCR derived clones were thensequenced to identify those that are bonafide cadherin sequences. Suchcandidate clones were then used as probes in Northern blot analysisusing synoviocyte RNA.

Cadherins have not been reported to be expressed in synoviocytes priorto the present invention. The present invention is based, at least inpart, on the discovery that synovial derived fibroblast-like cells andsynovial membrane lining cells express a cadherin. The gene encodingthis cadherin was cloned, monoclonal antibodies were made against it andits expression on transfectant cells, cultured human synoviocytes and infreshly isolated human rheumatoid synovium was demonstrated.

Example 1 Material and Methods

Antibodies. The β-catenin antisera was previously described (Cepek K L.et al. Proc Natl Acad Sci USA 93:6567-71, 1996). The pan-cadherinantiserum directed against the C-terminal 24 amino acids of chickenN-cadherin was previously described (Geiger B. et al. J Cell Sci97:607-14, 1990) and obtained from Sigma (St. Louis, Mo.). The specificmouse mAbs against human E-cadherin (E4.6, IgG₁) was raised in thislaboratory (Cepek K L. et al. Nature 372:190-3, 1994), the anti-humanN-cadherin (13A9, IgG₁) was kindly provided by M. Wheelock (Departmentof Biology, Toledo University, Toledo Ohio), the anti-human VE-cadherin(BV9, IgG₁) was a gift from M. G. Lampugnani (Laboratory of VascularBiology, Mario Negri Institute, Milano, Italy) and the anti-humanP-cadherin (NCC-CAD-299, IgG₁) was provided by S. Hirohashi (PathologyDivision, National Cancer Research Institute, Tokyo, Japan), Leu 4(anti-CD3) from Becton Dickinson (Mountain View, Calif.), OKT4(anti-CD4), OKT8 (anti-CD8) from Ortho Pharmaceutical Corp. (Raritan,N.J.), and anti-CD68 from Dako (Carpenteira, Calif.).

Cell Culture. The synovial membranes from RA patients diagnosed based oncurrent criteria (Arnett F C. et al. Arthritis Rheum 31:315-324, 1988)were obtained during hand and wrist synovectomy and joint replacementsurgical procedures. Synovial tissue was prepared by mincing and treatedwith 1 mg/ml collagenase (type 1, Worthington Biochemicals, Frehold,N.J.), 0.15 mg/ml Dnase I (Sigma, St. Louis, Mo.) and 5 nM CaCl₂ inphosphate buffer saline (PBS) solution (Gibco, Grand Island, N.Y.), androcked at 37° C. for 1 hr. The cell suspension was passed through a 40mesh metal sieve and placed in a 75 cm² tissue culture flask (BDLabware, Lincoln Park, N.J.). The cells released from the synovialtissue were plated in flasks in DME supplemented with 10% fetal calf and10% human serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mlpenicillin, 100 μg/ml streptomycin sulfate, and 50 μM 2-mercaptoethanoland maintained in 10% CO₂. Confluent monolayers were found to becomposed mainly of the type II (fibroblast like) synoviocytes after thethird passage.

The murine fibroblast L-cell line (ATCC CCL1.3) was grown in DMEM, highglucose with 10% bovine calf serum (Hyclone), 10 mM HEPES, 2 mML-glutamine, 1 mM sodium pyruvate, 10 μM non-essential amino acids(Gibco BRL, Gaithersburg, Md.), 100 U/ml penicillin, 100 μg/mlstreptomycin sulfate, and 50 μM 2-mercaptoethanol and maintained in 10%CO₂. L-cell transfectants were grown in the above medium with G418(Gibco) at 1 mg/ml.

Human embryonic kidney HEK293 cells (obtained from ATCC) were maintainedin 10% (vol/vol) heat-inactivated FBS (Hyclone Labs Inc., Logan, Utah)and DME medium (Gibco BRL) at 37° C. in 10% CO₂.

Cos-7 cells were grown in DMEM, high glucose with 10% Nu-Serum(Collaborative Research, Inc., Bedford, Mass.), 10 mM HEPES, 2 mML-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/mlstreptomycin sulfate, and 50 μM 2-mercaptoethanol and maintained in 10%CO₂.

Human breast epithelial 16E6.A5 cells were maintained as described(Cepek K L. et al. Proc Natl Acad Sci USA 93:6567-71, 1996).

The T lymphocytes were isolated from Ficoll-Hypaque density gradientderived PMBC using anti-CD3 (or anti-CD2 mAb) and magnetic beads. Theymay be used directly after release using Detach-a-bead or stimulatedwith PHA and cultured in IL-2 containing media as long term T celllines. The isolated fresh lymphocytes will be used in adhesion assays tosynovial cell monolayers. Adhesion will be examined in staticcell-to-cell assays in 96 well plates using fluorescently labeledlymphocytes. The percentage of fluorescently labeled T cells that bindto synovial cell monolayers is determined using a fluorescence platereader, and the blocking effects of specific mAb can be determined.

Labeling and Immunoprecipitation. 1×10⁷ confluent synoviocyte monolayerswere surface labeled with 2 mCi Na ¹²⁵I (Du Pont-New England Nuclear,Boston, Mass.) using lactoperoxidase and hydrogen peroxide in 0.5 ml PBSas previously described (Brenner, et al. 1987). The cells weresolubilized in lysis buffer containing Tris buffered saline (TBS, 50 mMTris-base, pH 7.6, 140 mM NaCl) with 1% Triton X-100, 8 mM iodoacetamide(IAA) and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis,Mo.) 1 mM CaCl₂ for 1 hr at 4° C. Detergent insoluble material wasremoved by centrifugation for 20 minutes at 8,000 g at 4° C. Thesupernatant was precleared with 6 μl normal rabbit serum and 300 μl187.1 mAb as culture supernatant for 30 minutes followed by two rounds(1 hr and 12 hrs) of 200 μl of a 10% (wt/vol) cell suspension of fixedStaphylococcus aureus Cowen strain I (PANSCORBIN, Calbiochem, San Diego,Calif.). Lysates containing the equivalent of 2×10⁶ cells wereimmunoprecipitated with 10-15 μl of rabbit antiserum or 15 μl of anti-Pcatenin antisera or 1 μl of E4.6 ascites, and 100 μl each of 13A9, BV9,Cad-299, 2G4, 5H6, 3H10 mAb supernatants plus 100 μl rat anti-mouseK-chain mAb 187.1 culture supernatant for optimal protein A binding.Immune complexes were then incubated with protein A-Sepharose (PharmaciaBiotechnology Inc., Piscataway, N.J.) for 1 hr at 4° C. with rocking.The immunoprecipitates were washed three times with 0.5% (vol/vol)Triton X-100 in 0.5 M NaCl with 50 mM, 1 mM CaCl₂. The resin pellet wasboiled in sample buffer (10% glycerol, 3% SDS, 0.5 M Tris, pH 6.8)containing 2-mercaptoethanol (5% final concentration, reducingconditions) and analyzed by 7.5% SDS-PAGE as described (Laemmli UK.Nature 227:680-5, 1970) and subjected to standard fluorographicprocedures.

Molecular Cloning of The Gene Encoding the Synovial Cadherin. A numberof new cadherin cDNA clones have been isolated using PCR based consensusoligonucleotides corresponding to cytoplasmic domain sequences that arehighly conserved among cadherins (Suzuki S. et al. Cell Reg 2:261-70,1991). Based upon alignments of the human cadherins, four regions ofidentity can be appreciated, corresponding to human E-cadherin residues753-762 (EEGGGEEDQD) (SEQ ID NO:3), residues 840-847 (SLSSLNSS) (SEQ IDNO:4), residues 853-859 (QDYDYLN) (SEQ ID NO:5), and residues 865-875(FKKLADMYGGG) (SEQ ID NO:6), which in each case are identical in E-, P-,and N-cadherins. Using these regions we designed the followingdegenerate oligonucleotides sense: 5′-GCGGGATCCGAIGARGGIGGNGGNGA-3′ (SEQID NO:7) and antisense: 5′GGGGAGCTCTCIGCIARYTTYTTRAA-3′ (N=A,T,G,C;I=Inosine; Y=C,T and R=A,G) (SEQ ID NO:8). mRNA was extracted fromrheumatoid synoviocytes and reversed transcribed and used as a templatefor PCR amplification using 0.5 U/reaction of Taq polymerase and thefollowing conditions: denaturing 95° C. for 30 sec, annealing 60° C. for1 minute, extension 72° C. for 1 minute during 30 cycles. The PCRproducts of the expected 385 bp size were cloned into pCRII plasmidusing the TA cloning system (Invitrogen Corp., Carlsbad, Calif.) andsubsequently sequenced with the Sequenase kit (United Stated BiochemicalCorp., Cleveland, Ohio) to identify those that appear to be bonafidecadherin sequences. We searched GenBank database using the FASTA/BLASTprogram.

Northern Blot. To confirm the expression of the cloned cadherin insynoviocytes we performed northern blot analysis. mRNA isolated fromsynoviocytes type B, 16A5 cells, Jurkat cells and a SKNSH werehybridized with the 385 bp fragment of the cadherin cloned by PCR andglyceraldehyde-3-phosphate dehydrogenase, as the control, both of whichwere labeled with [³²P] dCTP/dGTP by random priming. Hybridization wasperformed at 43° C. during 16 hrs. After hybridization, the membrane waswashed at a final stringency of 0.1×SSC with 2% (w/v) SDS at 56° C. andautoradiographed on Kodak MS film at −70° C.

Construction of Full Length Clone. Full length cadherin-11 wasconstructed by PCR using the following primers XV14(5′-CCAAAAATGAAGGAGAACTACT-3′) (SEQ ID NO:9) and XV15(5′-GGGGGATCCATTGTTAAGAATCGTCATCAAA-3′) (SEQ ID NO:10) comprising thecoding region of cadherin-11. The product of ˜2.4 kb was subcloned intothe Hind III/Bam H1 sites of pBluescript SK and sequenced to confirmthat no mutations were introduced during the PCR.

Construction of Cadherin-11-Fc Expression Vector. A double stranded DNAadapter containing a 5′ Msc I blunt end, and the final five codons ofthe human cadherin-11 extracellular region, and a 3′ Xho I cohesive endwas produced by annealing the complimentary oligonucleotides XVCad11A(5′-GCTGGCACCGTGGTTGGGAGAGT-3′) (SEQ ID NO:11) and XVCad11 E(5′-GGGGGGCTCGAGGTAGGCCTCTGCGTTGCAGG-3′) (SEQ ID NO:12) using PyrococcusFuriosus (PFU) (Stratagene, La Jolla, Calif.) according to themanufacturer's recommendations. This adapter was then ligated to the3′-end of an Hind III-Msc I fragment encoding the rest of theextracellular region of human cadherin-11 from the cadherin-11 fulllength clone generated previously. The resulting Hind III-Xho I fragmentwas introduced in frame, upstream of coding for the hinge and Fc regionof human IgG₁ in a derivative of pCDM8 (pCDM8Fc) also cleaved with HindIII-Xho I. The sequence of the junctional region is shown in FIG. 3. Theconstruct was sequenced to confirm its integrity at the junctionalregion using an automated DNA sequencer (Perkin Elmer). Finally, thecadherin-11-Fc cDNA was excised from pCDM8 using Hind III and Not I andinserted into the expression vector pCEP4 (Invitrogen Corp. Carlsbad,Calif.) cleaved with Hind III and Not I.

Production of Cadherin-11-Fc Proteins. HEK293 cells (10⁶ cells per 75cm² flask) were stably transfected with 20 μg plasmid DNA using theMammalian transfection kit (Stratagene). After growth for 24 hrs innonselective medium, the cells were transferred to 96-well tissueculture plates and incubated in selective medium containing 200 μg/mlhygromycin B. After 15 days, supernatants from wells containingresistant colonies were assayed for fusion proteins by ELISA.

To produce the cadherin-11-Fc protein, positive clones were grown intriple-layer 500-cm² flasks (Nunc, Roskilde, Denmark) in 10% (v/v)ultralow Ig FBS (GIBCO BRL), 200 μg/ml hygromycin B and DMEM. After 7-10days of culture, the supernatant was harvested and filtered through a0.2 μm membrane. The cadherin-11-Fc fusion protein was then purified ona previously unused GammaBind G-Sepharose column (Pharmacia Biotech,Piscataway, N.J.). The column was washed with TBS and 1 mM CaCl₂, pH7.4, and then eluted with 0.2 M glycine and 1 mM CaCl₂, pH 2.3.Fractions containing purified fusion protein were dialyzed into TBS and1 mM CaCl₂, pH 7.4 and then stored at −20° C. The purity of the fusionprotein was assessed by SDS-PAGE and Coomassie blue staining, and theconcentration was determined by Bradford assay using BSA as the standard(BioRad Labs., Hercules, Calif.).

Generation of L-cell-Cadherin-11 Stable Transfectants. To produce cellsexpressing cadherin-11 in the surface, L-cells were transfected with 20μg pLK/Cad11 or with the pLK neo vector alone using the Mammaliantransfection kit (Stratagene). Then transfected cells were selected byculture in 1 mg/ml G418.

Generation Of Mouse Monoclonal Antibodies Against Cadherin-11. The mAb2G4, 5H6 and 3H10 were produced by immunizing BALB/c mice with threeintraperitoneal injections of 20 μg of purified cadherin-11-Fc. Theinitial injection was in CFA and the subsequent 2 were in IFA at 2 weeksintervals, followed by a final boost IV of 30 μg. Three days after theintravenous immunization, splenocytes were isolated and fused with NS1murine myeloma cells in the presence of PEG (MW 1450) as describedpreviously (Lerner E A. Yale J Biol Med 54:387-402. 1981). Hybridomaswere selected with aminopterin-containing medium, and hybridomasupernatants were screened by differential ELISA in plates coated with301-Fc, E-cad-Fc and human IgG₁. Subsequent screening was done in theselected clones by FACS in cadherin-11 expressing cells in comparison toE-cadherin positive cells. The selected hybridomas were subcloned thriceby limiting dilution. The isotypes of 2G4 (IgG₁), 5H6 (IgG₁) and 3H10(IgG₁) were determined by ELISA using murine isotype specific mAb(Jackson Immunoresearch Lab).

Immunohistochemistry. Normal human tissue samples were obtained andsnap-frozen in OCT compound (Ames Co., Elkhart, Ind.) cooled by liquidnitrogen to about −140° C. Frozen tissue sections were sectioned (6 μm)with a CM 10800 cryostat (Leica Inc. Deerfield, Ill.), and then fixed inacetone for 10 minutes, briefly air-dried, and stained by an indirectimmunoperoxidase method using avidin-biotin-peroxidase complex (VectorLabs, Burlingame, Calif.) and 3-amino-9-ethylcarbazole (Sigma, St.Louis, Mo.) as the chromogen.

Results

Cloning of the Synoviocyte Cadherin. The PCR based degenerateoligonucleotide cloning of a cadherin in type B synoviocyte cDNA showeda specific product in synoviocytes that was absent in the negativecontrol (FIG. 1). This product was identified as a fragment ofcadherin-11 when sequenced and compared to the GenBank database. We thenconfirmed the expression of cadherin-11 in synovial derived RNA byNorthern blot using as a probe the 385 bp cloned by PCR. FIG. 2 showsthe positive hybridization of cad-11 probe in synoviocytes type B and ina positive control, a neuroblastoma cell line (SKNSH) and was absent inepithelial cells (16EA5) and Jurkat cells. In order to obtain thefull-length gene we generated a clone comprising the entire codingsequence of human cadherin-11 by PCR. The PCR product obtained wassequenced to confirm lack of mutations.

Production of Human Cadherin-11-Fc Fusion Protein. A construct encodingthe extracellular portion of human cadherin-11 was linked in frame to aconstruct encoding the Fc region of human IgG₁ (including the hinge,C_(H)2, and C_(H)3 domains). Transfection of HEK293 cells and selectionwith hygromycin B led to the generation of stable lines expressingsoluble cadherin-11-Fc fusion protein. The cadherin-11-Fc fusion proteinwas expected to be dimeric due to the presence of disulfide bonds in the(FIG. 3), possibly similar to cadherin dimers on the cell surface(Shapiro L. et al. Nature 374:327-37, 1995; Nagar B M. et al. 380:360-4,1996). After purification on protein G-Sepharose, SDS-PAGE revealed thepresence of a protein of the expected (˜123 kD) size after reduction.

Generation of Anti-Cadherin-11 Monoclonal Antibodies. To produce mAbwhich specifically recognized the extracellular region of cadherin-11,BALB/c mice were immunized with the purified cadherin-11-Fc fusionprotein. mAb which preferentially reacted with cadherin-11-Fc but nothuman IgG₁ nor E-cadherin-Fc by ELISA were selected for further study.Three of these mAb antibodies 3H10, 5H6 and 2G4 recognized specificallycadherin-11-Fc in ELISA. Also these mAb stain cadherin-11 expressed onthe surface of L-cells transfected with full length cadherin-11 as shownin FIG. 4. These three mAb also precipitated a protein of 123 kD fromthe surface-labeled synoviocytes.

In order to study the pattern of tissue distribution of the protein weperformed indirect immunohistochemistry in frozen tissue sections. Wenoted the remarkable staining pattern in which anti-cadherin-11 mAbpreferentially stained the lining cells in RA synovium. This suggeststhat cadherin-11 plays an important role in determining the adhesion ofthe synovial lining cells to one another and thereby determining thetissue architecture of the synovium. This role may be critical to thegrowth and proliferation as well as to the activation of the synovialmembrane cells. Note that synovial membranes lack an epithelial layerand instead have the synovial lining layer. Interestingly theseanti-cadherin-11 mAb also recognized few cells in the sublining adjacentto the T-cell areas infiltrating the synovium (FIG. 6, panels A and B).

Example 2 Adhesion Assays

Monolayers of adherent cells (i.e., human synoviocytes) were grown inflat bottomed 96-well Linbro tissue culture plates. 10⁴ adherent cellsin 100 μl complete media were added per well and allowed to grow for twoto three days until they reached confluence. Just prior to the additionof T cells or cadherin-11 counter-receptor transfected COS 7 cells, theadherent cell monolayers were washed with assay media. To label T cellsor COS-7 cells, 25 μg of 2′,7′-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF-AM, Molecular Probes, Inc., Eugene, Oreg.)was diluted in 5 μl DMSO and added to a suspension of 5×10⁶/ml T cellsor COS-7 cells in complete media. The cells were incubated at 37° C. for25 minutes then washed twice in assay media (PBS containing 1 mM CaCl₂,2 mM MgCl₂ and 10 mM HEPES). After washing, 50,000 labeled T cells orCOS-7 cells in 100 μl of assay media were added to the adherent cellmonolayers. T cells or COS-7 cells were allowed to settle onto adherentcell monolayers for 25 or 40 minutes at 37° C. Unbound cells wereremoved by flicking media from the plate. Bound cells were detectedusing a Fluorescence plate reader (IDEXX Co., Portland, Me.). Ifantibody blocking was performed, the T cells, COS-7 cells, orsynoviocyte cell monolayers were pre-incubated with a 1:250 dilution ofascites fluid or 10 μg/ml of purified mAb for five minutes at 37° C.prior to encounter with the second cell type. At least four replicatesare performed. The % cells bound is calculated by reading thefluorescence units obtained after unbound cells were washed off,dividing this number by the input fluorescence units and multiplying by100. Serial dilutions of labeled cells showed that as few as 1000 cellsare detected in the linear range.

Example 3 An Adhesion Assay for Selecting Library Members asPharmaceutical Lead Compounds

The adhesion assay described herein is based upon the assay described byCepek, K., et al., in J. Immunol. 150(8):3459-3470 (1993), the entirecontents of which are incorporated herein by reference.

To screen a molecular library or other mixture for the presence of afunctionally equivalent peptide analog or a library member capable ofinhibiting cadherin-11 mediated adhesion, T cells are washed with HBSS(Hanks buffered saline solution Gibco) and pre-equilibrated with HBSScontaining serial dilutions of the library or other peptide-containingor small molecule-containing solution (over a broad concentration range(e.g., 1 ng/ml to 100 ug/ml) for selected times (e.g., 30 min, 1 hour, 2hours, 6 hours) at 37° C. before incubation with synovial monolayersthat have been washed with HBSS. Functionally equivalent peptide analogsor cadherin-11 inhibitory agents are identified by their ability toinhibit the binding of T cells to the synovial monolayer.

Example 4 Cadherin-11 Counter-Receptor on T and B Cells

Flow cytometric analysis of the synovial T cell line 5 and CP-B cellswith anti-cadherin-11 monoclonal antibody 2G4 was performed. Controlstaining was carried out with the control monoclonal antibody P3. FIG. 6demonstrates the lack of staining of either the T or the B cell linewith the cadherin-11 specific monoclonal antibody.

To test the binding of cadherin-11 to T and B cells, the synovial T cellline 5 and CP-B cells were used. Plates were treated with cadherin-11-Fcor ICAM-1-Fc and IgG1. Both cell lines were labeled with a fluorescentdye and were added to the plates and input fluorescence was determinedin a fluorescence plate reader. Cells were allow to adhere for 30minutes at 37° C., subsequently washed and the ratio of bound cells tofluorescence was determined. The percentage of cells bound wasdetermined using the equation: fluorescence after wash/inputfluorescence×100. The binding to cadherin-11-Fc but not to controlproteins ICAM-1-Fc and IgG1 is blocked by anti-cadherin-11 monoclonalantibody 7D3. The results are expressed as the mean +1 SD (n=4) as shownin FIG. 7.

Several lines of evidence support the possibility that cadherin-11 bindsto a counter-receptor expressed on B and T lymphocytes. First,immunohistochemical analysis of RA. synovium demonstrated cadherin-11expression in sublining cells in close contact with lymphocytes. Second,some T cell lines derived from RA synovium and B cells derived from theperipheral blood bind specifically to cadherin-11-Fc and thisinteraction is blocked by anti-cadherin-11 mAbs. Third, cadherin-11 wasnot expressed on these leukocyte lines, suggesting that the interactionis not mediated by homophilic but by a heterophilic binding to acadherin-11 receptor (C11CR) expressed on B and T cells.

Discussion

Here we described for the first time the presence of a cadherinexpressed on synoviocytes. Cadherins are expressed on all cells thatform solid tissues and are responsible for segregating and sorting cellsduring tissue morphogenesis. They play a role in establishing cellpolarity and maintaining tissue morphology in adult tissues. Althoughcadherins function classically to mediate homophilic cell-to-celladhesion, they sometimes bind to nonidentical cadherins or to integrins,such as integrin α^(E)β₇.

Our findings of preferential expression of cadherin-11 in the lining ofRA synovium suggest that this adhesion molecule could be the keymolecule used by the invasive pannus to attach to cartilage andeventually erode into bone, particularly since this cadherin has beenreported to be expressed by osteoblasts. This is of particular relevancebecause we can interfere with the chronic destructive processcharacteristic of RA if we can modulate the adhesive function ofcadherin-11.

We consider the identification of cadherin-11 from human type Bsynoviocytes an important finding and its tissue distribution within theRA synovium supports its relevant role in invading and eventuallyeroding the adjacent bone.

It should be understood that the preceding is merely a detaileddescription of certain preferred embodiments. It therefore should beapparent to those of ordinary skill in the art that variousmodifications and equivalents can be made without departing from thespirit and scope of the invention. It is intended that the inventionencompass all such modifications within the scope of the appendedclaims.

All references, patents and patent applications and publications thatare recited in this application are incorporated in their entiretyherein by reference.

1. A method for treating a subject having an inflammatory joint disordercomprising administering to a subject in need of such treatment atherapeutically effective amount of a cadherin-11 inhibitory nucleicacid molecule that hybridizes under stringent conditions to a nucleicacid molecule having the sequence of SEQ ID NO: 1 and that inhibitsexpression of cadherin-11 protein, wherein the cadherin-11 inhibitorynucleic acid molecule inhibits binding of cadherin-11 to a cadherin-11counter-receptor.
 2. The method of claim 1, wherein the inflammatoryjoint disorder is chronic synovitis.
 3. The method of claim 1, whereinthe inflammatory joint disorder is an autoimmune disease.
 4. The methodof claim 3, wherein the autoimmune disease is rheumatoid arthritis. 5.The method of claim 1, wherein the cadherin-11 inhibitory nucleic acidmolecule is administered locally to a synovium of the subject.
 6. Themethod of claim 1, wherein the cadherin-11 inhibitory nucleic acidmolecule is an antisense nucleic acid molecule.
 7. The method of claim1, wherein the cadherin-11 inhibitory nucleic acid molecule inhibitstranslation of cadherin-11 mRNA.
 8. The method of claim 7, wherein thecadherin-11 inhibitory nucleic acid molecule is an oligoribonucleotide.9. The method of claim 8, wherein the cadherin-11 inhibitory nucleicacid molecule is 20-30 bases in length.
 10. The method of claim 1,wherein the cadherin-11 inhibitory nucleic acid molecule is introducedinto a synoviocyte of the subject.